Methods for treating lung disease

ABSTRACT

The methods and assays described herein relate to the diagnosis, prognosis, and treatment of subjects with emphysema, COPD, and/or cigarette-induced lung damage. In some embodiments, the methods and assays relate to subjects with a decreased level of NLRX1 expression. In some embodiments, the methods and assays relate to the administration of an agonist of NLRX1 and/or an inhibitor of MAVS.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims benefit under 35 U.S.C. §119(e) of U.S.Provisional Application No. 62/130,811 filed Mar. 10, 2015, the contentsof which are incorporated herein by reference in their entirety.

GOVERNMENT SUPPORT

This invention was made with federal funding under Grant Nos.R56HL119511, HL-079328, and PO1 HL114501 awarded by the NationalInstitutes of Health. The U.S. government has certain rights in theinvention.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Feb. 4, 2016, isnamed 058040-084261-US_SL.txt and is 34,231 bytes in size.

TECHNICAL FIELD

The technology described herein relates to the diagnosis, prognosis, andtreatment of lung disease, e.g. COPD, emphysema, and/or cigarettesmoke-induced lung damage.

BACKGROUND

Cigarette smoke causes a broad spectrum of diseases characterized byinflammation and tissue remodeling, including chronic obstructivepulmonary disease (COPD). In many of these disorders the inflammation isbelieved to drive disease pathogenesis. This can be seen in COPD, thefourth leading cause of death in the world, where pulmonary inflammationis believed to be causally related to the emphysema and other pathologicalterations in the lungs from these patients.

Cigarette smoke activates caspase 1 and IL-18, thereby regulating theinflammasome. However, the mechanism(s) that controls lung inflammasomeactivation at baseline and the alterations that cigarette smoke inducesto activate the inflammasome have not been defined.

SUMMARY

MAVS (mitochondrial antiviral signaling molecule) regulates inflammatoryand remodeling responses in viral infections and is inhibited in thehealthy, non-infected lung. It is demonstrated herein that cigarettesmoke causes damage to lung tissue, at least in part, by inhibitingNLRX1, which inhibits MAVS in healthy tissue. Thus, cigarette smokecauses an increase in the activity of MAVS, resulting in inflammationand pathological tissue remodeling. Restoration of NLRX1 in lung exposedto cigarette smoke reserved the pathological effects of cigarette smoke.Accordingly, provided herein are methods of diagnosing, prognosing, andtreating COPD, emphysema, and/or cigarette smoke-induced lung damagerelating to the discovery that NLRX1 is suppressed in diseased tissues.

In one aspect, described herein is a method of treating COPD in asubject in need thereof, the method comprising administering aninhibitor of MAVS. In one aspect, described herein is a method oftreating cigarette smoke-induced lung damage in a subject in needthereof, the method comprising administering an inhibitor of MAVS. Insome embodiments, the cigarette smoke-induced lung damage is selectedfrom the group consisting of: inflammation; alveolar destruction;protease induction; structural cell apoptosis; and inflammasomeactivation. In one aspect, described herein is a method of treatingemphysema in a subject in need thereof, the method comprisingadministering an inhibitor of MAVS. In some embodiments, the emphysemais cigarette smoke induced emphysema.

In some embodiments, the inhibitor of MAVS is an agonist of NLRX1. Insome embodiments, the inhibitor of MAVS is a modulator ofNLRX1-dependent pathways. In some embodiments, the agonist of NLRX1 is anucleic acid encoding NLRX1 or or an NLRX1 polypeptide.

In one aspect, described herein is a method of treating COPD in asubject in need thereof, the method comprising administering aninhibitor of MAVS.

In one aspect, described herein is a method of treating cigarettesmoke-induced lung damage in a subject in need thereof, the methodcomprising administering an agonist of NLRX1. In some embodiments, thecigarette smoke-induced lung damage is selected from the groupconsisting of: inflammation; alveolar destruction; protease induction;structural cell apoptosis; and inflammasome activation. In one aspect,described herein is a method of treating emphysema in a subject in needthereof, the method comprising administering an agonist of NLRX1. Insome embodiments, the emphysema is cigarette smoke induced emphysema.

In some embodiments, the subject is a subject determined to have adecreased level of NLRX1 expression.

In one aspect, described herein is an assay comprising: measuring thelevel of NLRX1 in a test sample obtained from a subject; wherein andecrease in the expression level relative to a reference level indicatesthe subject has a higher risk of having or developing COPD, emphysema,and/or cigarette-induced lung damage. In one aspect, described herein isa method of identifying a subject in need of treatment for COPD,emphysema, and/or cigarette-induced lung damage, the method comprising:measuring the level of NLRX1 in a test sample obtained from a subject;and identifying the subject as being in need of treatment for COPD,emphysema, and/or cigarette-induced lung damage when the expressionlevel of NLRX1 is decreased relative to a reference level. In oneaspect, described herein is a method of determining if a subject is atrisk for COPD, emphysema, and/or cigarette-induced lung damage, themethod comprising: measuring the level of NLRX1 in a test sampleobtained from a subject; comparing the level of NLRX1 in the sample to areference level of NLRX1; determining that the subject is at risk forCOPD, emphysema, and/or cigarette-induced lung damage when the level ofNLRX1 is decreased relative to a reference level; and determining thatthe subject is not at risk for COPD, emphysema, and/or cigarette-inducedlung damage when the level of NLRX1 is not decreased relative to areference level. In one aspect, described herein is a method ofdetermining the efficacy of a treatment for COPD, emphysema, and/orcigarette-induced lung damage, the method comprising: (a) measuring thelevel of NLRX1 in a test sample obtained from a subject beforeadministration of the treatment; (b) measuring the level of NLRX1 in atest sample obtained from a subject after administration of thetreatment; (c) determining that the treatment is efficacious when theexpression level determined in step (b) is not decreased relative to theexpression level determined in step (a); and (d) determining that thetreatment is not efficacious when the expression level determined instep (b) is decreased relative to the expression level determined instep (a). In one aspect, described herein is a method of treatment forCOPD, emphysema, and/or cigarette-induced lung damage comprising;measuring the level of NLRX1 in a test sample obtained from a subject;treating the subject when the level of NLRX1 is decreased relative to areference level. In one aspect, described herein is a method oftreatment for COPD, emphysema, and/or cigarette-induced lung damagecomprising; treating a subject determined to have a level of NLRX1 whichis decreased relative to a reference level. In some embodiments, thetreatment comprises a treatment selected from the group consisting of:administration of a bronchodilator; administration of an inhaledsteroid; administration of oxygen therapy; bullectomy; lung volumereduction surgery; smoking cessation; lung transplant; administration ofan inhibitor of MAVS; administration of an agonist of NLRX1;administration of a nucleic acid encoding NLRX1; and administration ofan NLRX1 polypeptide.

In some embodiments, the level of NLRX1 is determined by measuring thelevel of a nucleic acid. In some embodiments, the level of NLRX1 isdetermined by measuring the level of NLRX1 RNA transcript. In someembodiments, the level of the nucleic acid is determined using a methodselected from the group consisting of: RT-PCR; quantitative RT-PCR;Northern blot; microarray based expression analysis; next-generationsequencing; and RNA in situ hybridization. In some embodiments, thelevel of NLRX1 is determined by measuring the level of NLRX1polypeptide. In some embodiments, the level of the polypeptide isdetermined using a method selected from the group consisting of Westernblot; immunoprecipitation; enzyme-linked immunosorbent assay (ELISA);radioimmunological assay (MA); sandwich assay; fluorescence in situhybridization (FISH); immunohistological staining; radioimmunometricassay; immunofluoresence assay; mass spectroscopy; FACS; andimmunoelectrophoresis assay. In some embodiments, the polypeptide levelis measured using immunochemistry. In some embodiments, the antibodyreagent is detectably labeled or generates a detectable signal.

In some embodiments, the expression level of NLRX1 is normalizedrelative to the expression level of one or more reference genes orreference proteins. In some embodiments, the reference level of NLRX1 isthe expression level of NLRX1 in a prior sample obtained from thesubject. In some embodiments, the subject is a subject with a history ofsmoking. In some embodiments, the sample is selected from the groupconsisting of: a lung biopsy; bronchoalveolar lavage (BAL); sputum;induced sputum; blood; plasma; and serum. In some embodiments, themethod or assay further comprises the step of treating the subject witha treatment selected from the group consisting of: administration of abronchodilator; administration of an inhaled steroid; administration ofoxygen therapy; bullectomy; lung volume reduction surgery; smokingcessation; lung transplant; administration of an inhibitor of MAVS;administration of an agonist of NLRX1; administration of a nucleic acidencoding NLRX1; and administration of an NLRX1 polypeptide.

In one aspect, described herein is a kit for performing the method orassay of the foregoing aspects.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1H demonstrate NLRX1 mRNA suppression in patients with COPD andits correlation with disease severity. The levels of NLRX1 mRNA in LTRCsamples were plotted in controls (0) and in patients with COPD ofvarying severity (GOLD 1, 2, 3, and 4) (FIG. 1A). In FIG. 1B the levelsof NLRX1 are compared in no disease (controls, 0) and patients with,mild to moderate (GOLD 1 and 2) and severe COPD (GOLD 3 and 4). Thelevels of NLRX1 protein in mitochondria-enriched tissue fractions (MF)from controls (0) and GOLD 1, 2, 3, and 4 individuals were evaluated bywestern blot analysis (FIG. 1C). The levels of NLRX1 protein in thesewestern evaluations were evaluated by densitometry and compared to thelevels of expression of the voltage dependent anion channel (VDAC) (**p<0.01). The correlations between the levels of NLRX1 mRNA in the LTRCsamples and pre-bronchodilator FEV₁ (% of predicted value) (FIG. 1D) andpost-bronchodilator FEV₁ (% of predicted value) (FIG. 1E) are alsoillustrated. FIG. 1F depicts is a box-whiskers plot from the Pittsburghcohort describing the relationship between NLRX1 gene expression inmicroarray evaluations and radiologic emphysema (** p<0.05 compared tocontrol and the >40% emphysema group). From the Asan cohort, thestandardized levels of NLRX1 gene transcriptome were plotted in controlsand in patients with COPD (FIG. 1G). In FIG. 1H the levels of NLRX1 geneexpression are compared in no disease (controls) and patients with COPDof varying severity (GOLD 1, 2, and 3). The bars in FIGS. 1A, 1B, and 1Grepresent the mean±SEM of the noted evaluations. The statistics thatwere applied were the nonparametric Kruskal-Wallis test (FIG. 1A),Mann-Whitney U test (FIGS. 1B and 1C), Pearson correlation analysis(FIGS. 1D and 1E), two-tailed unpaired t-test (FIGS. 1F and 1G) andANOVA test (FIG. 1H). In FIGS. 1F and 1H, the box shows the mean andstandard deviation of the group and the whiskers show minimum andmaximum expression values.

FIGS. 2A-2M demonstrate that cigarette smoke (CS)-induced suppression ofNLRX1 and the role of NLRX1 in CS-induced pulmonary inflammation andalveolar destruction. NLRX1 mRNA (FIG. 2A) and protein (FIG. 2B) aresuppressed by CS exposure. In FIG. 2B, the bar graph to the right of thegel represents the results of densitometry comparing the levels of NLRX1and voltage dependent anion channel (VDAC) in mitochondria-enrichedfractions (MF) and cytosol-enriched fractions (CF). After 3 months of CSexposure, western blot analysis of the NLRX1 protein expression in BALcells are presented in FIG. 2C. After 3 months of CS exposure, the rolesof NLRX1 were evaluated using comparisons of responses in wild type (WT)and NLRX1 null mice. BAL total cell recovery (FIG. 2D), representativehistology (FIG. 2E), lung morphometry (FIG. 2F), the expression ofMMP-12 mRNA (FIG. 2G), the expression of interferon (IFN)-alpha (α) 4mRNA, and TUNEL evaluation of structural (epithelial and endothelial)cells (FIG. 2I) are illustrated. The levels of active IL-1β (FIG. 2J)and IL-18 (FIG. 2K) in lung lysates were also measured by ELISA. Inpanel (FIG. 2L), western blot evaluations of lung tissue lysates areused to characterize the activation status of IL-1β, IL-18 andcaspase-1. In FIG. 2M, the measurement of mean chord lengh is presentedfrom lentiviral NLRX1 overexpressed mice (NLRX1 vector+) and appropriatecontrols (control vector+) after 6 month-CS-exposure. Size bar on FIG.2E represents 400 μm. The values in FIGS. 2A, 2D, 2F-2K and 2M representthe mean±SEM of evaluations in a minimum of 5 mice (* p<0.05, **p<0.01). FIGS. 2B, 2C, and 2L are representative of a minimum of 3similar experiments.

FIGS. 3A-3K demonstrate the role of MAVS in CS-induced pulmonaryinflammation and alveolar destruction. After 6 months of CS exposure,the roles of MAVS were evaluated using comparisons of responses in WTand MAVS null mice. BAL total cell recovery (FIG. 3A), representativehistology (FIG. 3B), lung morphometry (FIG. 3C) and the expression ofMMP-12 mRNA (FIG. 3D) are illustrated. In FIG. 3E, western blotevaluations of lung tissue lysates were used to demonstrate theactivation status of IL-18 and caspase-1. The results of TUNELevaluations of structural cells (FIG. 3F), the expression of interferon(IFN)-α4 mRNA (FIG. 3G) and the expression of NLRX1 mRNA (FIG. 3H) arealso illustrated. The results of the evaluation of mean chord length(FIG. 3I), TUNEL evaluations of structural cells (FIG. 3J) and themeasurement of the level of IL-18 in lung tissues (FIG. 3K) arepresented from MAVS^(−/−)/NLRX1^(−/−) mice, NLRX1^(−/−) mice and wildtype (MAVS^(+/+)/NLRX1^(+/+)) controls after 3 month-CS-exposure. Sizebar on FIG. 3B represents 400 μm. The values in FIGS. 3A, 3C, 3D, and3F-3K represent the mean±SEM of evaluations in a minimum of 5 mice. (*p<0.05, ** p<0.01). FIG. 3E is representative of a minimum of 3 similarevaluations.

FIGS. 4A-4B demonstrate the correlations between the levels of (FIG. 4A)NLRX1 mRNA and pre-bronchodilator FEV₁ (% of predicted value) and (FIG.4B) NLRX1 mRNA and post-bronchodilator FEV₁ (% of predicted value),respectively, in the Korean Asan cohort

FIG. 5 demonstrates the statistical result of the comparison betweeneach subgroup in Korean Asan cohort. Because only one patient was in thesubgroup of GOLD3, no comparison was undertaken with the subgroup ofGOLD 3 (* For the overall comparison between all subgroups, please seeFIG. 1H).

FIG. 6 demonstrates the levels of NLRX1 mRNA and the status of smokinghistory in the Korean Asan cohort.

FIGS. 7A-7B demonstrate the levels of (FIG. 7A) MAVS mRNA and (FIG. 7B)protein in LTRC samples were plotted in controls (0) and in patientswith COPD of varying severity (GOLD 1, 2, 3, and 4).

FIGS. 8A-8C demonstrate the correlations between the levels of NLRX1mRNA and (FIG. 8A) diffusing capacity (DLCO), (FIG. 8B) BODE index, and(FIG. 8C) BORG scale, respectively, in the LTRC cohort.

FIG. 9 demonstrates the levels of CXCL13 protein in LTRC samples plottedin controls (0) and in patients with COPD of varying severity (GOLD 1,2, 3, and 4)

FIGS. 10A-10D demonstrate the correlations between the levels of CXCL13protein and (FIG. 10A) post-bronchodilator FEV₁ (% of predicted value),(FIG. 10B) NLRX1 mRNA, (FIG. 10C) BODE index, and (FIG. 10D) SGRQ score,respectively, in LTRC cohort.

FIGS. 11A-11B demonstrate, from the Westernblot presented on FIG. 2C thedensitometric evaluation of (FIG. 11A) NLRX1 expression and (FIG. 11B)MAVS expression, respectively, are demonstrated (** p<0.01).

FIG. 12 demonstrates the suppression of NLRX1 mRNA accumulation in3-month cigarette smoke-exposed lungs (CS+) and its recovery 3 monthsafter the cessation of CS exposure (#) (** p<0.01).

FIG. 13 demonstrates the enhancement of CXCL13 mRNA expression after3-month cigarette smoke-exposure (CS+) in the lungs from NLRX1 deficient(NLRX1−/−) mice compared to those from WT controls (NLRX1+/+) (* p<0.05,** p<0.01).

FIG. 14 demonstrates the restoration of CS-induced emphysematousalveolar destruction after NLRX1 gene overexpression in vivo. After6-month-CS-exposure, morphometric evaluation of alveolar surface areawas analyzed (* p<0.05, ** p<0.01).

FIG. 15 depicts measurement of alveolar surface area after3-month-CS-exposure in wild type (WT) controls, NLRX1^(−/−) andNLRX1^(−/−)/MAVS^(−/−) mice (** p<0.01).

FIG. 16 demonstrates the enhancement of CXCL13 mRNA expression after3-month cigarette smoke-exposure (CS+) in the lungs from NLRX1 deficient(NLRX1−/−) mice compared to those from WT controls (NLRX1+/+) (* p<0.05,** p<0.01).

FIG. 17 is a diagram of an exemplary embodiment of a system forperforming an assay for determining the level of NLRX1 in a sampleobtained from a subject.

FIG. 18 is a diagram of an exemplary embodiment of a comparison moduleas described herein.

FIG. 19 is a diagram of an exemplary embodiment of an operating systemand applications for a computing system as described herein.

DETAILED DESCRIPTION

As described herein, the inventors have found that inhibition of MAVS,e.g., by stimulating NLRX1 activity, has a therapeutic effect on lungdiseases, e.g. COPD, emphysema, and/or cigarette smoke-induced lungdamage. Accordingly, provided herein are methods of treating COPD,emphysema, and/or cigarette smoke-induced lung damage by administeringan inhibitor of MAVS and/or an agonist of NLRX1.

As used herein, “emphysema” refers to a chronic lung disease whichaffects the alveoli and/or the ends of the smallest bronchi. Thecondition is characterized by destructive changes and enlargement of thealveoli (air sacs) within the lungs. The lung loses its elasticity andtherefore these areas of the lungs become enlarged. These enlarged areastrap stale air and do not effectively exchange it with fresh air. Thisresults in difficult breathing and may result in insufficient oxygenbeing delivered to the blood. The predominant symptom in patients withemphysema is shortness of breath. In some embodiments, the emphysema canbe cigarette-smoke induced emphysema. As used herein, “COPD” or “chronicobstructive pulmonary disease” is generally applied to chronicrespiratory disease processes characterized by the persistentobstruction of bronchial air flow. COPD patients can suffer fromconditions such as bronchitis, cystic fibrosis, asthma or emphysema. Asused herein, “cigarette smoke-induced lung damage” refers to damage tolung tissues occurring in subjects exposed to cigarette smoke, includingbut not limited to: inflammation; alveolar destruction; proteaseinduction; structural cell apoptosis; and inflammasome activation.Cigarette smoke-induced lung damage can be present in a subject havingor diagnosed as having emphysema or COPD or in a subject not having ornot diagnosed as having emphysema or COPD. In some embodiments of any ofthe aspects described herein, the patient can be a patient with ahistory of smoking.

As used herein, “MAVS” or “mitochondrial antiviral-signaling protein”refers to a protein found in the mitochondria, that when aggregated,activates IRF3 dimerization, contributing to the recognition of virii.MAVS is also known in the art as VISA, IPS-1, and Cardif. Sequences forMAVS expression products are known for a number of species, e.g., humanMAVS (NCBI Gene ID: 57506) mRNA (SEQ ID NO: 3; NCBI Ref Seq: NM_020746)and polypeptide (SEQ ID NO: 4; NCBI Ref Seq: NP_065797).

As used herein, “inhibitor” refers to an agent which can decrease theexpression and/or activity of the targeted expression product (e.g. mRNAencoding the target or a target polypeptide), e.g. by at least 10% ormore, e.g. by 10% or more, 50% or more, 70% or more, 80% or more, 90% ormore, 95% or more, or 98% or more. The efficacy of an inhibitor of, forexample, MAVS, e.g. its ability to decrease the level and/or activity ofMAVS can be determined, e.g. by measuring the level of an expressionproduct of MAVS and/or the activity of MAVS. Methods for measuring thelevel of a given mRNA and/or polypeptide are known to one of skill inthe art, e.g. RTPCR with primers can be used to determine the level ofRNA and Western blotting with an antibody (e.g. an anti-MAVS antibody,e.g. Cat No. ab189109 Abcam; Cambridge, Mass.) can be used to determinethe level of a polypeptide. The activity of, e.g. MAVS can be determinedusing methods known in the art and described elsewhere herein, e.g., bymeasuring the levels of expression of CXCL13, MMP-12, cathepsisn K,cathepsin S, type 1 IFNs, caspase 1, IL-1β, and/or IL-18, wheredecreased MAVS levels and/or activity results in decreased levels ofCXCL13, MMP-12, cathepsisn K, cathepsin S, type 1 IFNs, caspase 1,IL-1β, and/or IL-18. In some embodiments, the inhibitor can be aninhibitory nucleic acid; an aptamer; an antibody reagent; an antibody;or a small molecule.

In some embodiments, an inhibitor of MAVS can be an agonist of NLRX1. Insome embodiments, an inhibitor of MAVS can be a modulator ofNLRX1-dependent pathways.

As used herein, “NLRX1” or “nucleotide-binding oligomerization domainleucine rich repeat containing X1” refers to an intracellular signalingprotein with a N-terminal mitochondrion localization signal, a NACHTdomain, an a C-terminal LRR. NLRX1 is also known in the art as NODS,NODS, and CLR11.3. Sequences for NLRX1 expression products are known fora number of species, e.g., human NLRX1 (NCBI Gene ID: 79671) mRNA (SEQID NO: 1; NCBI Ref Seq: NM_001282144) and polypeptide (SEQ ID NO: 2;NCBI Ref Seq: NP_001269073).

As used herein, “agonist” refers to any agent that increases the leveland/or activity of the target, e.g, of NLRX1. As used herein, the term“agonist” refers to an agent which increases the expression and/oractivity of the target by at least 10% or more, e.g. by 10% or more, 50%or more, 100% or more, 200% or more, 500% or more, or 1000% or more. Theefficacy of an agonist of, for example, NLRX1, e.g. its ability todecrease the level and/or activity of NLRX1 can be determined, e.g. bymeasuring the level of an expression product of NLRX1 and/or theactivity of NLRX1. Methods for measuring the level of a given mRNAand/or polypeptide are known to one of skill in the art, e.g. RTPCR withprimers can be used to determine the level of RNA and Western blottingwith an antibody (e.g. an anti-NLRX1 antibody, e.g. Cat No. ab105412Abcam; Cambridge, Mass.) can be used to determine the level of apolypeptide. The activity of, e.g. NLRX1 can be determined using methodsknown in the art and described elsewhere herein, e.g., by measuring thelevels of expression of CXCL13, MMP-12, cathepsisn K, cathepsin S, type1 IFNs, caspase 1, IL-1β, and/or IL-18, where increased NLRX1 levelsand/or activity results in decreased levels of CXCL13, MMP-12,cathepsisn K, cathepsin S, type 1 IFNs, caspase 1, IL-1β, and/or IL-18.Non-limiting examples of agonists of NLRX1 can include NLRX1polypeptides or fragments thereof and nucleic acids encoding a NLRX1polypeptide, e.g. a polypeptide comprising the sequence SEQ ID NO: 2 ora nucleic acid comprising the sequence of SEQ ID NO: 1 or variantsthereof. In some embodiments, the agonist of NRLX1 can be an NLRX1polypeptide. In some embodiments, the agonist of NLRX1 can be anengineered and/or recombinant polypeptide. In some embodiments, theagonist of NLRX1 can be a nucleic acid encoding NLRX1, e.g. a functionalfragment thereof.

In some embodiments, the agonist of NLRX1 can be a modulator of anNLRX1-dependent pathway. NLRX1-dependent pathways are known in the artand described, e.g., in Allen et al. Front Immunol 2014 5:169; which isincorporated by reference herein in its entirety. By way of non-limitingexample, a modulator of an NLRX1-dependent pathway suitable for use inthe methods described herein can be an inhibitor of TRAF6, IKK-gamma,IKK-alpha/beta, IκB, p65, p50, RIG1, TBK1, IRF, IFN-1, and/or IL-6.

In some embodiments, a therapy as described herein, e.g. an inhibitor ofMAVS and/or an agonist of NLRX1 can be targeted to the mitochondria.Targeting can be achieved, e.g. by conjugating the agent to a targetinggroup or including the agent in a composition comprising a targetinggroup (e.g. a nanoparticle). Targeting groups can include, e.g., a cellor tissue targeting agent, e.g., a lectin, glycoprotein, lipid orprotein, e.g., an antibody, that binds to a specified cell type such aslung cell, among others, or a cell permeation agent. Non-limitingexamples of mitochondrial targeting groups can includetriphenylphosphonium (TPP); mitochondrial targeting sequencepolypeptides, and the like. Mitochondrial targeting is further describedin the art, see, e.g., Weinberg and Chandel Nat Chem Biol 2015 11:9-15;Dongworth et al. Future Cardiol 2014 10:255-272; and Sureshbabu FrontPhysiol 2013 4:384; each of which is incorporated by reference herein inits entirety.

As described herein, decreased levels of NLRX1 expression and/oractivity indicate that a subject has an increased risk of having and/ordeveloping COPD, emphysema, and/or cigarette smoke-induced lung damage.Accordingly, in some embodiments of any of the aspects described herein,the subject that is treated in accordance with the methods describedherein is a subject having or identified as having a decreased level ofNLRX1 expression and/or activity.

In one aspect, described herein is an assay comprising: measuring thelevel of NLRX1 in a test sample obtained from a subject; wherein andecrease in the expression level relative to a reference level indicatesthe subject has a higher risk of having or developing COPD, emphysema,and/or cigarette-induced lung damage. In one aspect, described herein isa method of identifying a subject in need of treatment for COPD,emphysema, and/or cigarette-induced lung damage, the method comprising:measuring the level of NLRX1 in a test sample obtained from a subject;and identifying the subject as being in need of treatment for COPD,emphysema, and/or cigarette-induced lung damage when the expressionlevel of NLRX1 is decreased relative to a reference level. In oneaspect, described herein is a method of determining if a subject is atrisk for COPD, emphysema, and/or cigarette-induced lung damage, themethod comprising: measuring the level of NLRX1 in a test sampleobtained from a subject; comparing the level of NLRX1 in the sample to areference level of NLRX1; determining that the subject is at risk forCOPD, emphysema, and/or cigarette-induced lung damage when the level ofNLRX1 is decreased relative to a reference level; and determining thatthe subject is not at risk for COPD, emphysema, and/or cigarette-inducedlung damage when the level of NLRX1 is not decreased relative to areference level. In some embodiments, the level of NLRX1 is decreasedrelative to a reference amount if it is less than the reference amountby a statistically significant amount.

In some embodiments, measurement of the level of a target, e.g. of anNLRX1 expression product can comprise a transformation. As used herein,the term “transforming” or “transformation” refers to changing an objector a substance, e.g., biological sample, nucleic acid or protein, intoanother substance. The transformation can be physical, biological orchemical. Exemplary physical transformation includes, but not limitedto, pre-treatment of a biological sample, e.g., from whole blood toblood serum by differential centrifugation. A biological/chemicaltransformation can involve at least one enzyme and/or a chemical reagentin a reaction. For example, a DNA sample can be digested into fragmentsby one or more restriction enzyme, or an exogenous molecule can beattached to a fragmented DNA sample with a ligase. In some embodiments,a DNA sample can undergo enzymatic replication, e.g., by polymerasechain reaction (PCR).

Transformation, measurement, and/or detection of a target molecule, e.g.a NLRX1 mRNA or polypeptide can comprise contacting a sample obtainedfrom a subject with a reagent (e.g. a detection reagent) which isspecific for the target, e.g., a NLRX1-specific reagent. In someembodiments, the target-specific reagent is detectably labeled. In someembodiments, the target-specific reagent is capable of generating adetectable signal. In some embodiments, the target-specific reagentgenerates a detectable signal when the target molecule is present.

Methods to measure NLRX1 gene expression products are well known to askilled artisan. Such methods to measure gene expression products, e.g.,protein level, include ELISA (enzyme linked immunosorbent assay),western blot, immunoprecipitation, and immunofluorescence usingdetection reagents such as an antibody or protein binding agents.Alternatively, a peptide can be detected in a subject by introducinginto a subject a labeled anti-peptide antibody and other types ofdetection agent. For example, the antibody can be labeled with adetectable marker whose presence and location in the subject is detectedby standard imaging techniques.

For example, antibodies for NLRX1 are commercially available and can beused for the purposes of the invention to measure protein expressionlevels, e.g. anti-NLRX1 (Cat. No. ab105412; Abcam, Cambridge Mass.).Alternatively, since the amino acid sequences for NRLX1 are known andpublically available at NCBI website, one of skill in the art can raisetheir own antibodies against these polypeptides of interest for thepurpose of the invention.

The amino acid sequences of the polypeptides described herein, e.g.NLRX1 have been assigned NCBI accession numbers for different speciessuch as human, mouse and rat. In particular, the NCBI accession numbersfor the amino acid sequence of human NLRX1 is included herein, e.g. SEQID NO: 2.

In some embodiments, immunohistochemistry (“IHC”) andimmunocytochemistry (“ICC”) techniques can be used. IHC is theapplication of immunochemistry to tissue sections, whereas ICC is theapplication of immunochemistry to cells or tissue imprints after theyhave undergone specific cytological preparations such as, for example,liquid-based preparations. Immunochemistry is a family of techniquesbased on the use of an antibody, wherein the antibodies are used tospecifically target molecules inside or on the surface of cells. Theantibody typically contains a marker that will undergo a biochemicalreaction, and thereby experience a change of color, upon encounteringthe targeted molecules. In some instances, signal amplification can beintegrated into the particular protocol, wherein a secondary antibody,that includes the marker stain or marker signal, follows the applicationof a primary specific antibody.

In some embodiments, the assay can be a Western blot analysis.Alternatively, proteins can be separated by two-dimensional gelelectrophoresis systems. Two-dimensional gel electrophoresis is wellknown in the art and typically involves iso-electric focusing along afirst dimension followed by SDS-PAGE electrophoresis along a seconddimension. These methods also require a considerable amount of cellularmaterial. The analysis of 2D SDS-PAGE gels can be performed bydetermining the intensity of protein spots on the gel, or can beperformed using immune detection. In other embodiments, protein samplesare analyzed by mass spectroscopy.

Immunological tests can be used with the methods and assays describedherein and include, for example, competitive and non-competitive assaysystems using techniques such as Western blots, radioimmunoassay (RIA),ELISA (enzyme linked immunosorbent assay), “sandwich” immunoassays,immunoprecipitation assays, immunodiffusion assays, agglutinationassays, e.g. latex agglutination, complement-fixation assays,immunoradiometric assays, fluorescent immunoassays, e.g. FIA(fluorescence-linked immunoassay), chemiluminescence immunoassays(CLIA), electrochemiluminescence immunoassay (ECLIA, countingimmunoassay (CIA), lateral flow tests or immunoassay (LFIA), magneticimmunoassay (MIA), and protein A immunoassays. Methods for performingsuch assays are known in the art, provided an appropriate antibodyreagent is available. In some embodiment, the immunoassay can be aquantitative or a semi-quantitative immunoassay.

An immunoassay is a biochemical test that measures the concentration ofa substance in a biological sample, typically a fluid sample such asurine, using the interaction of an antibody or antibodies to itsantigen. The assay takes advantage of the highly specific binding of anantibody with its antigen. For the methods and assays described herein,specific binding of the target polypeptides with respective proteins orprotein fragments, or an isolated peptide, or a fusion protein describedherein occurs in the immunoassay to form a target protein/peptidecomplex. The complex is then detected by a variety of methods known inthe art. An immunoassay also often involves the use of a detectionantibody.

Enzyme-linked immunosorbent assay, also called ELISA, enzyme immunoassayor EIA, is a biochemical technique used mainly in immunology to detectthe presence of an antibody or an antigen in a sample. The ELISA hasbeen used as a diagnostic tool in medicine and plant pathology, as wellas a quality control check in various industries.

In one embodiment, an ELISA involving at least one antibody withspecificity for the particular desired antigen (e.g., NLRX1 as describedherein) can also be performed. A known amount of sample and/or antigenis immobilized on a solid support (usually a polystyrene micro titerplate). Immobilization can be either non-specific (e.g., by adsorptionto the surface) or specific (e.g. where another antibody immobilized onthe surface is used to capture antigen or a primary antibody). After theantigen is immobilized, the detection antibody is added, forming acomplex with the antigen. The detection antibody can be covalentlylinked to an enzyme, or can itself be detected by a secondary antibodywhich is linked to an enzyme through bio-conjugation. Between each stepthe plate is typically washed with a mild detergent solution to removeany proteins or antibodies that are not specifically bound. After thefinal wash step the plate is developed by adding an enzymatic substrateto produce a visible signal, which indicates the quantity of antigen inthe sample. Older ELISAs utilize chromogenic substrates, though newerassays employ fluorogenic substrates with much higher sensitivity.

In another embodiment, a competitive ELISA is used. Purified antibodiesthat are directed against a target polypeptide or fragment thereof arecoated on the solid phase of multi-well plate, i.e., conjugated to asolid surface. A second batch of purified antibodies that are notconjugated on any solid support is also needed. These non-conjugatedpurified antibodies are labeled for detection purposes, for example,labeled with horseradish peroxidase to produce a detectable signal. Asample (e.g., a blood sample) from a subject is mixed with a knownamount of desired antigen (e.g., a known volume or concentration of asample comprising a target polypeptide) together with the horseradishperoxidase labeled antibodies and the mixture is then are added tocoated wells to form competitive combination. After incubation, if thepolypeptide level is high in the sample, a complex of labeled antibodyreagent-antigen will form. This complex is free in solution and can bewashed away. Washing the wells will remove the complex. Then the wellsare incubated with TMB (3, 3′, 5, 5′-tetramethylbenzidene) colordevelopment substrate for localization of horseradishperoxidase-conjugated antibodies in the wells. There will be no colorchange or little color change if the target polypeptide level is high inthe sample. If there is little or no target polypeptide present in thesample, a different complex in formed, the complex of solid supportbound antibody reagents-target polypeptide. This complex is immobilizedon the plate and is not washed away in the wash step. Subsequentincubation with TMB will produce much color change. Such a competitiveELSA test is specific, sensitive, reproducible and easy to operate.

There are other different forms of ELISA, which are well known to thoseskilled in the art. The standard techniques known in the art for ELISAare described in “Methods in Immunodiagnosis”, 2nd Edition, Rose andBigazzi, eds. John Wiley & Sons, 1980; and Oellerich, M. 1984, J. Clin.Chem. Clin. Biochem. 22:895-904. These references are herebyincorporated by reference in their entirety.

In one embodiment, the levels of a polypeptide in a sample can bedetected by a lateral flow immunoassay test (LFIA), also known as theimmunochromatographic assay, or strip test. LFIAs are a simple deviceintended to detect the presence (or absence) of antigen, e.g. apolypeptide, in a fluid sample. There are currently many LFIA tests areused for medical diagnostics either for home testing, point of caretesting, or laboratory use. LFIA tests are a form of immunoassay inwhich the test sample flows along a solid substrate via capillaryaction. After the sample is applied to the test strip it encounters acolored reagent (generally comprising antibody specific for the testtarget antigen) bound to microparticles which mixes with the sample andtransits the substrate encountering lines or zones which have beenpretreated with another antibody or antigen. Depending upon the level oftarget polypeptides present in the sample the colored reagent can becaptured and become bound at the test line or zone. LFIAs areessentially immunoassays adapted to operate along a single axis to suitthe test strip format or a dipstick format. Strip tests are extremelyversatile and can be easily modified by one skilled in the art fordetecting an enormous range of antigens from fluid samples such asurine, blood, water, and/or homogenized tissue samples etc. Strip testsare also known as dip stick test, the name bearing from the literalaction of “dipping” the test strip into a fluid sample to be tested.LFIA strip tests are easy to use, require minimum training and caneasily be included as components of point-of-care test (POCT)diagnostics to be use on site in the field. LFIA tests can be operatedas either competitive or sandwich assays. Sandwich LFIAs are similar tosandwich ELISA. The sample first encounters colored particles which arelabeled with antibodies raised to the target antigen. The test line willalso contain antibodies to the same target, although it may bind to adifferent epitope on the antigen. The test line will show as a coloredband in positive samples. In some embodiments, the lateral flowimmunoassay can be a double antibody sandwich assay, a competitiveassay, a quantitative assay or variations thereof. Competitive LFIAs aresimilar to competitive ELISA. The sample first encounters coloredparticles which are labeled with the target antigen or an analogue. Thetest line contains antibodies to the target/its analogue. Unlabelledantigen in the sample will block the binding sites on the antibodiespreventing uptake of the colored particles. The test line will show as acolored band in negative samples. There are a number of variations onlateral flow technology. It is also possible to apply multiple capturezones to create a multiplex test.

The use of “dip sticks” or LFIA test strips and other solid supportshave been described in the art in the context of an immunoassay for anumber of antigen biomarkers. U.S. Pat. Nos. 4,943,522; 6,485,982;6,187,598; 5,770,460; 5,622,871; 6,565,808, U.S. patent application Ser.No. 10/278,676; U.S. Ser. No. 09/579,673 and U.S. Ser. No. 10/717,082,which are incorporated herein by reference in their entirety, arenon-limiting examples of such lateral flow test devices. Examples ofpatents that describe the use of “dip stick” technology to detectsoluble antigens via immunochemical assays include, but are not limitedto U.S. Pat. Nos. 4,444,880; 4,305,924; and 4,135,884; which areincorporated by reference herein in their entireties. The apparatusesand methods of these three patents broadly describe a first componentfixed to a solid surface on a “dip stick” which is exposed to a solutioncontaining a soluble antigen that binds to the component fixed upon the“dip stick,” prior to detection of the component-antigen complex uponthe stick. It is within the skill of one in the art to modify theteachings of this “dip stick” technology for the detection ofpolypeptides using antibody reagents as described herein.

Other techniques can be used to detect the level of a polypeptide in asample. One such technique is the dot blot, and adaptation of Westernblotting (Towbin et at., Proc. Nat. Acad. Sci. 76:4350 (1979)). In aWestern blot, the polypeptide or fragment thereof can be dissociatedwith detergents and heat, and separated on an SDS-PAGE gel before beingtransferred to a solid support, such as a nitrocellulose or PVDFmembrane. The membrane is incubated with an antibody reagent specificfor the target polypeptide or a fragment thereof. The membrane is thenwashed to remove unbound proteins and proteins with non-specificbinding. Detectably labeled enzyme-linked secondary or detectionantibodies can then be used to detect and assess the amount ofpolypeptide in the sample tested. The intensity of the signal from thedetectable label corresponds to the amount of enzyme present, andtherefore the amount of polypeptide. Levels can be quantified, forexample by densitometry.

In some embodiments, the level of, e.g., NLRX1, can be measured, by wayof non-limiting example, by Western blot; immunoprecipitation;enzyme-linked immunosorbent assay (ELISA); radioimmunological assay(RIA); sandwich assay; fluorescence in situ hybridization (FISH);immunohistological staining; radioimmunometric assay; immunofluoresenceassay; mass spectroscopy and/or immunoelectrophoresis assay.

In certain embodiments, the gene expression products as described hereincan be instead determined by determining the level of messenger RNA(mRNA) expression of the genes described herein, e.g. NLRX1. Suchmolecules can be isolated, derived, or amplified from a biologicalsample, such as a blood sample. Techniques for the detection of mRNAexpression is known by persons skilled in the art, and can include butnot limited to, PCR procedures, RT-PCR, quantitative RT-PCR Northernblot analysis, differential gene expression, RNA protection assay,microarray based analysis, next-generation sequencing; hybridizationmethods, etc.

In general, the PCR procedure describes a method of gene amplificationwhich is comprised of (i) sequence-specific hybridization of primers tospecific genes or sequences within a nucleic acid sample or library,(ii) subsequent amplification involving multiple rounds of annealing,elongation, and denaturation using a thermostable DNA polymerase, and(iii) screening the PCR products for a band of the correct size. Theprimers used are oligonucleotides of sufficient length and appropriatesequence to provide initiation of polymerization, i.e. each primer isspecifically designed to be complementary to a strand of the genomiclocus to be amplified. In an alternative embodiment, mRNA level of geneexpression products described herein can be determined byreverse-transcription (RT) PCR and by quantitative RT-PCR (QRT-PCR) orreal-time PCR methods. Methods of RT-PCR and QRT-PCR are well known inthe art.

In some embodiments, the level of an mRNA can be measured by aquantitative sequencing technology, e.g. a quantitative next-generationsequence technology. Methods of sequencing a nucleic acid sequence arewell known in the art. Briefly, a sample obtained from a subject can becontacted with one or more primers which specifically hybridize to asingle-strand nucleic acid sequence flanking the target gene sequenceand a complementary strand is synthesized. In some next-generationtechnologies, an adaptor (double or single-stranded) is ligated tonucleic acid molecules in the sample and synthesis proceeds from theadaptor or adaptor compatible primers. In some third-generationtechnologies, the sequence can be determined, e.g. by determining thelocation and pattern of the hybridization of probes, or measuring one ormore characteristics of a single molecule as it passes through a sensor(e.g. the modulation of an electrical field as a nucleic acid moleculepasses through a nanopore). Exemplary methods of sequencing include, butare not limited to, Sanger sequencing, dideoxy chain termination,high-throughput sequencing, next generation sequencing, 454 sequencing,SOLiD sequencing, polony sequencing, Illumina sequencing, Ion Torrentsequencing, sequencing by hybridization, nanopore sequencing, Helioscopesequencing, single molecule real time sequencing, RNAP sequencing, andthe like. Methods and protocols for performing these sequencing methodsare known in the art, see, e.g. “Next Generation Genome Sequencing” Ed.Michal Janitz, Wiley-VCH; “High-Throughput Next Generation Sequencing”Eds. Kwon and Ricke, Humanna Press, 2011; and Sambrook et al., MolecularCloning: A Laboratory Manual (4 ed.), Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y., USA (2012); which are incorporated byreference herein in their entireties.

The nucleic acid sequences of the genes described herein, e.g., NLRX1,have been assigned NCBI accession numbers for different species such ashuman, mouse and rat. For example, the human NRLX1 mRNA (e.g. SEQ IDNO: 1) is known. Accordingly, a skilled artisan can design anappropriate primer based on the known sequence for determining the mRNAlevel of the respective gene.

Nucleic acid and ribonucleic acid (RNA) molecules can be isolated from aparticular biological sample using any of a number of procedures, whichare well-known in the art, the particular isolation procedure chosenbeing appropriate for the particular biological sample. For example,freeze-thaw and alkaline lysis procedures can be useful for obtainingnucleic acid molecules from solid materials; heat and alkaline lysisprocedures can be useful for obtaining nucleic acid molecules fromurine; and proteinase K extraction can be used to obtain nucleic acidfrom blood (Roiff, A et al. PCR: Clinical Diagnostics and Research,Springer (1994)).

In some embodiments, one or more of the reagents (e.g. an antibodyreagent and/or nucleic acid probe) described herein can comprise adetectable label and/or comprise the ability to generate a detectablesignal (e.g. by catalyzing reaction converting a compound to adetectable product). Detectable labels can comprise, for example, alight-absorbing dye, a fluorescent dye, or a radioactive label.Detectable labels, methods of detecting them, and methods ofincorporating them into reagents (e.g. antibodies and nucleic acidprobes) are well known in the art.

In some embodiments, detectable labels can include labels that can bedetected by spectroscopic, photochemical, biochemical, immunochemical,electromagnetic, radiochemical, or chemical means, such as fluorescence,chemifluoresence, or chemiluminescence, or any other appropriate means.The detectable labels used in the methods described herein can beprimary labels (where the label comprises a moiety that is directlydetectable or that produces a directly detectable moiety) or secondarylabels (where the detectable label binds to another moiety to produce adetectable signal, e.g., as is common in immunological labeling usingsecondary and tertiary antibodies). The detectable label can be linkedby covalent or non-covalent means to the reagent. Alternatively, adetectable label can be linked such as by directly labeling a moleculethat achieves binding to the reagent via a ligand-receptor binding pairarrangement or other such specific recognition molecules. Detectablelabels can include, but are not limited to radioisotopes, bioluminescentcompounds, chromophores, antibodies, chemiluminescent compounds,fluorescent compounds, metal chelates, and enzymes.

In other embodiments, the detection reagent is label with a fluorescentcompound. When the fluorescently labeled reagent is exposed to light ofthe proper wavelength, its presence can then be detected due tofluorescence. In some embodiments, a detectable label can be afluorescent dye molecule, or fluorophore including, but not limited tofluorescein, phycoerythrin, phycocyanin, o-phthaldehyde, fluorescamine,Cy3™, Cy5™, allophycocyanine, Texas Red, peridenin chlorophyll, cyanine,tandem conjugates such as phycoerythrin-Cy5™, green fluorescent protein,rhodamine, fluorescein isothiocyanate (FITC) and Oregon Green™,rhodamine and derivatives (e.g., Texas red and tetrarhodimineisothiocynate (TRITC)), biotin, phycoerythrin, AMCA, CyDyes™,6-carboxyfhiorescein (commonly known by the abbreviations FAM and F),6-carboxy-2′,4′,7′,4,7-hexachlorofiuorescein (HEX),6-carboxy-4′,5′-dichloro-2′,7′-dimethoxyfiuorescein (JOE or J),N,N,N′,N′-tetramethyl-6carboxyrhodamine (TAMRA or T),6-carboxy-X-rhodamine (ROX or R), 5-carboxyrhodamine-6G (R6G5 or G5),6-carboxyrhodamine-6G (R6G6 or G6), and rhodamine 110; cyanine dyes,e.g. Cy3, Cy5 and Cy7 dyes; coumarins, e.g umbelliferone; benzimidedyes, e.g. Hoechst 33258; phenanthridine dyes, e.g. Texas Red; ethidiumdyes; acridine dyes; carbazole dyes; phenoxazine dyes; porphyrin dyes;polymethine dyes, e.g. cyanine dyes such as Cy3, Cy5, etc; BODIPY dyesand quinoline dyes. In some embodiments, a detectable label can be aradiolabel including, but not limited to ³H, ¹²⁵I, ³⁵S, ¹⁴C, ³²P, and³³P. In some embodiments, a detectable label can be an enzyme including,but not limited to horseradish peroxidase and alkaline phosphatase. Anenzymatic label can produce, for example, a chemiluminescent signal, acolor signal, or a fluorescent signal. Enzymes contemplated for use todetectably label an antibody reagent include, but are not limited to,malate dehydrogenase, staphylococcal nuclease, delta-V-steroidisomerase, yeast alcohol dehydrogenase, alpha-glycerophosphatedehydrogenase, triose phosphate isomerase, horseradish peroxidase,alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase,ribonuclease, urease, catalase, glucose-VI-phosphate dehydrogenase,glucoamylase and acetylcholinesterase. In some embodiments, a detectablelabel is a chemiluminescent label, including, but not limited tolucigenin, luminol, luciferin, isoluminol, theromatic acridinium ester,imidazole, acridinium salt and oxalate ester. In some embodiments, adetectable label can be a spectral colorimetric label including, but notlimited to colloidal gold or colored glass or plastic (e.g.,polystyrene, polypropylene, and latex) beads.

In some embodiments, detection reagents can also be labeled with adetectable tag, such as c-Myc, HA, VSV-G, HSV, FLAG, V5, HIS, or biotin.Other detection systems can also be used, for example, abiotin-streptavidin system. In this system, the antibodiesimmunoreactive (i. e. specific for) with the biomarker of interest isbiotinylated. Quantity of biotinylated antibody bound to the biomarkeris determined using a streptavidin-peroxidase conjugate and achromagenic substrate. Such streptavidin peroxidase detection kits arecommercially available, e. g. from DAKO; Carpinteria, Calif. A reagentcan also be detectably labeled using fluorescence emitting metals suchas ¹⁵²Eu, or others of the lanthanide series. These metals can beattached to the reagent using such metal chelating groups asdiethylenetriaminepentaacetic acid (DTPA) or ethylenediaminetetraaceticacid (EDTA).

A level which is less than a reference level can be a level which isless by at least about 10%, at least about 20%, at least about 50%, atleast about 600%, at least about 80%, at least about 90%, or less thanthe reference level. In some embodiments, a level which is less than areference level can be a level which is statistically significantly lessthan the reference level. In some embodiments, the reference can be alevel of NLRX1 in a population of subjects who do not have or are notdiagnosed as having, and/or do not exhibit signs or symptoms of COPD,emphysema, and/or cigarette smoke-induced lung damage. In someembodiments, the reference can also be a level of expression of NLRX1 ina control sample, a pooled sample of control individuals or a numericvalue or range of values based on the same. In some embodiments, thereference can be the level of NLRX1 in a sample obtained from the samesubject at an earlier point in time, e.g., the methods described hereincan be used to determine if a subject's risk or likelihood of developingCOPD, emphysema, and/or cigarette smoke-induced lung damage isincreasing.

In some embodiments, the level of expression products of no more than200 other genes is determined. In some embodiments, the level ofexpression products of no more than 100 other genes is determined. Insome embodiments, the level of expression products of no more than 20other genes is determined. In some embodiments, the level of expressionproducts of no more than 10 other genes is determined.

In some embodiments of the foregoing aspects, the expression level of agiven gene, e.g., NLRX1, can be normalized relative to the expressionlevel of one or more reference genes or reference proteins.

The term “sample” or “test sample” as used herein denotes a sample takenor isolated from a biological organism, e.g., a blood or plasma samplefrom a subject. Exemplary biological samples include, but are notlimited to, a biofluid sample; serum; plasma; urine; saliva; and/ortissue sample etc. The term also includes a mixture of theabove-mentioned samples. The term “test sample” also includes untreatedor pretreated (or pre-processed) biological samples. In someembodiments, a test sample can comprise cells from subject. In someembodiments, the test sample can be a lung biopsy; bronchoalveolarlavage (BAL); sputum; induced sputum; blood; plasma; and serum.

The test sample can be obtained by removing a sample from a subject, butcan also be accomplished by using previously sample (e.g. isolated at aprior timepoint and isolated by the same or another person). Inaddition, the test sample can be freshly collected or a previouslycollected sample.

In some embodiments, the test sample can be an untreated test sample. Asused herein, the phrase “untreated test sample” refers to a test samplethat has not had any prior sample pre-treatment except for dilutionand/or suspension in a solution. Exemplary methods for treating a testsample include, but are not limited to, centrifugation, filtration,sonication, homogenization, heating, freezing and thawing, andcombinations thereof. In some embodiments, the test sample can be afrozen test sample, e.g., a frozen tissue. The frozen sample can bethawed before employing methods, assays and systems described herein.After thawing, a frozen sample can be centrifuged before being subjectedto methods, assays and systems described herein. In some embodiments,the test sample is a clarified test sample, for example, bycentrifugation and collection of a supernatant comprising the clarifiedtest sample. In some embodiments, a test sample can be a pre-processedtest sample, for example, supernatant or filtrate resulting from atreatment selected from the group consisting of centrifugation,filtration, thawing, purification, and any combinations thereof. In someembodiments, the test sample can be treated with a chemical and/orbiological reagent. Chemical and/or biological reagents can be employedto protect and/or maintain the stability of the sample, includingbiomolecules (e.g., nucleic acid and protein) therein, duringprocessing. One exemplary reagent is a protease inhibitor, which isgenerally used to protect or maintain the stability of protein duringprocessing. The skilled artisan is well aware of methods and processesappropriate for pre-processing of biological samples required fordetermination of the level of an expression product as described herein.

In some embodiments, the methods, assays, and systems described hereincan further comprise a step of obtaining a test sample from a subject.In some embodiments, the subject can be a human subject. In someembodiments, the subject can be a subject in need of treatment for (e.g.having or diagnosed as having) COPD, emphysema, and/or cigarettesmoke-induced lung damage.

In one aspect, described herein is a method of determining the efficacyof a treatment for COPD, emphysema, and/or cigarette-induced lungdamage, the method comprising: (a) measuring the level of NLRX1 in atest sample obtained from a subject before administration of thetreatment; (b) measuring the level of NLRX1 in a test sample obtainedfrom a subject after administration of the treatment; (c) determiningthat the treatment is efficacious when the expression level determinedin step (b) is not decreased relative to the expression level determinedin step (a); and (d) determining that the treatment is not efficaciouswhen the expression level determined in step (b) is decreased relativeto the expression level determined in step (a).

In one aspect, described herein is a method of treatment for COPD,emphysema, and/or cigarette-induced lung damage comprising; measuringthe level of NLRX1 in a test sample obtained from a subject; treatingthe subject when the level of NLRX1 is decreased relative to a referencelevel. In one aspect, described herein is a method of treatment forCOPD, emphysema, and/or cigarette-induced lung damage comprising;treating a subject determined to have a level of NLRX1 which isdecreased relative to a reference level. Treatments for COPD, emphysema,and cigarette smoke-induced lung damage are known in the art and caninclude, by way of non-limiting example administration of abronchodilator; administration of an inhaled steroid; administration ofoxygen therapy; bullectomy; lung volume reduction surgery; smokingcessation; and lung transplant. In some embodiments, a treatment forCOPD, emphysema, and/or cigarette smoke-induced lung damage can includeadministration of an inhibitor of MAVS; administration of an agonist ofNLRX1; administration of a nucleic acid encoding NLRX1; and/oradministration of an NLRX1 polypeptide.

In some embodiments, the methods described herein relate to treating asubject having or diagnosed as having, e.g., emphysema with an inhibitorof MAVS and/or an agonist of NLRX1. Subjects having emphysema can beidentified by a physician using current methods of diagnosing emphysema.Symptoms and/or complications of emphysema which characterize theseconditions and aid in diagnosis are well known in the art and includebut are not limited to, shortness of breath. Tests that may aid in adiagnosis of, e.g. emphysema include, but are not limited to, chestX-rays, CT scans, blood oxygenation tests, and lung function tests. Afamily history of emphysema, or exposure to risk factors for emphysema(e.g. smoking) can also aid in determining if a subject is likely tohave emphysema or in making a diagnosis of emphysema.

The compositions and methods described herein can be administered to asubject having or diagnosed as having COPD, emphysema, and/or cigarettesmoke-induced lung damage. In some embodiments, the methods describedherein comprise administering an effective amount of compositionsdescribed herein, e.g. an inhibitor of MAVS and/or an agonist of NLRX1to a subject in order to alleviate a symptom of COPD, emphysema, and/orcigarette smoke-induced lung damage. As used herein, “alleviating asymptom” of a disease or condition is ameliorating any condition orsymptom associated with the disease or condition. As compared with anequivalent untreated control, such reduction is by at least 5%, 10%,20%, 40%, 50%, 60%, 80%, 90%, 95%, 99% or more as measured by anystandard technique. A variety of means for administering thecompositions described herein to subjects are known to those of skill inthe art. Such methods can include, but are not limited to oral,parenteral, intravenous, intramuscular, subcutaneous, transdermal,airway (aerosol), pulmonary, cutaneous, topical, or injectionadministration. Administration can be local or systemic.

The term “effective amount” as used herein refers to the amount of aninhibitor of MAVS and/or an agonist of NLRX1 needed to alleviate atleast one or more symptom of the disease or disorder, and relates to asufficient amount of pharmacological composition to provide the desiredeffect. The term “therapeutically effective amount” therefore refers toan amount of an inhibitor of MAVS and/or an agonist of NLRX1 that issufficient to provide a particular effect when administered to a typicalsubject. An effective amount as used herein, in various contexts, wouldalso include an amount sufficient to delay the development of a symptomof the disease, alter the course of a symptom disease (for example butnot limited to, slowing the progression of a symptom of the disease), orreverse a symptom of the disease. Thus, it is not generally practicableto specify an exact “effective amount”. However, for any given case, anappropriate “effective amount” can be determined by one of ordinaryskill in the art using only routine experimentation.

Effective amounts, toxicity, and therapeutic efficacy can be determinedby standard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., for determining the LD50 (the dose lethal to 50% of thepopulation) and the ED50 (the dose therapeutically effective in 50% ofthe population). The dosage can vary depending upon the dosage formemployed and the route of administration utilized. The dose ratiobetween toxic and therapeutic effects is the therapeutic index and canbe expressed as the ratio LD50/ED50. Compositions and methods thatexhibit large therapeutic indices are preferred. A therapeuticallyeffective dose can be estimated initially from cell culture assays.Also, a dose can be formulated in animal models to achieve a circulatingplasma concentration range that includes the IC50 (i.e., theconcentration of an inhibitor of MAVS and/or an agonist of NLRX1, whichachieves a half-maximal inhibition of symptoms) as determined in cellculture, or in an appropriate animal model. Levels in plasma can bemeasured, for example, by high performance liquid chromatography. Theeffects of any particular dosage can be monitored by a suitablebioassay, e.g., assay for inflammation in the lungs, among others. Thedosage can be determined by a physician and adjusted, as necessary, tosuit observed effects of the treatment.

In some embodiments, the technology described herein relates to apharmaceutical composition comprising an inhibitor of MAVS and/or anagonist of NLRX1 as described herein, and optionally a pharmaceuticallyacceptable carrier. Pharmaceutically acceptable carriers and diluentsinclude saline, aqueous buffer solutions, solvents and/or dispersionmedia. The use of such carriers and diluents is well known in the art.Some non-limiting examples of materials which can serve aspharmaceutically-acceptable carriers include: (1) sugars, such aslactose, glucose and sucrose; (2) starches, such as corn starch andpotato starch; (3) cellulose, and its derivatives, such as sodiumcarboxymethyl cellulose, methylcellulose, ethyl cellulose,microcrystalline cellulose and cellulose acetate; (4) powderedtragacanth; (5) malt; (6) gelatin; (7) lubricating agents, such asmagnesium stearate, sodium lauryl sulfate and talc; (8) excipients, suchas cocoa butter and suppository waxes; (9) oils, such as peanut oil,cottonseed oil, safflower oil, sesame oil, olive oil, corn oil andsoybean oil; (10) glycols, such as propylene glycol; (11) polyols, suchas glycerin, sorbitol, mannitol and polyethylene glycol (PEG); (12)esters, such as ethyl oleate and ethyl laurate; (13) agar; (14)buffering agents, such as magnesium hydroxide and aluminum hydroxide;(15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18)Ringer's solution; (19) ethyl alcohol; (20) pH buffered solutions; (21)polyesters, polycarbonates and/or polyanhydrides; (22) bulking agents,such as polypeptides and amino acids (23) serum component, such as serumalbumin, HDL and LDL; (22) C₂-C₁₂ alcohols, such as ethanol; and (23)other non-toxic compatible substances employed in pharmaceuticalformulations. Wetting agents, coloring agents, release agents, coatingagents, sweetening agents, flavoring agents, perfuming agents,preservative and antioxidants can also be present in the formulation.The terms such as “excipient”, “carrier”, “pharmaceutically acceptablecarrier” or the like are used interchangeably herein. In someembodiments, the carrier inhibits the degradation of the active agent,e.g. an inhibitor of MAVS and/or an agonist of NLRX1 as describedherein.

In some embodiments, the pharmaceutical composition comprising aninhibitor of MAVS and/or an agonist of NLRX1 as described herein can bea parenteral dose form. Since administration of parenteral dosage formstypically bypasses the patient's natural defenses against contaminants,parenteral dosage forms are preferably sterile or capable of beingsterilized prior to administration to a patient. Examples of parenteraldosage forms include, but are not limited to, solutions ready forinjection, dry products ready to be dissolved or suspended in apharmaceutically acceptable vehicle for injection, suspensions ready forinjection, and emulsions. In addition, controlled-release parenteraldosage forms can be prepared for administration of a patient, including,but not limited to, DUROS®-type dosage forms and dose-dumping.

Suitable vehicles that can be used to provide parenteral dosage forms ofan inhibitor of MAVS and/or an agonist of NLRX1 as disclosed within arewell known to those skilled in the art. Examples include, withoutlimitation: sterile water; water for injection USP; saline solution;glucose solution; aqueous vehicles such as but not limited to, sodiumchloride injection, Ringer's injection, dextrose Injection, dextrose andsodium chloride injection, and lactated Ringer's injection;water-miscible vehicles such as, but not limited to, ethyl alcohol,polyethylene glycol, and propylene glycol; and non-aqueous vehicles suchas, but not limited to, corn oil, cottonseed oil, peanut oil, sesameoil, ethyl oleate, isopropyl myristate, and benzyl benzoate. Compoundsthat alter or modify the solubility of a pharmaceutically acceptablesalt of an inhibitor of MAVS and/or an agonist of NLRX1 as disclosedherein can also be incorporated into the parenteral dosage forms of thedisclosure, including conventional and controlled-release parenteraldosage forms.

Pharmaceutical compositions comprising an inhibitor of MAVS and/or anagonist of NLRX1 can also be formulated to be suitable for oraladministration, for example as discrete dosage forms, such as, but notlimited to, tablets (including without limitation scored or coatedtablets), pills, caplets, capsules, chewable tablets, powder packets,cachets, troches, wafers, aerosol sprays, or liquids, such as but notlimited to, syrups, elixirs, solutions or suspensions in an aqueousliquid, a non-aqueous liquid, an oil-in-water emulsion, or awater-in-oil emulsion. Such compositions contain a predetermined amountof the pharmaceutically acceptable salt of the disclosed compounds, andmay be prepared by methods of pharmacy well known to those skilled inthe art. See generally, Remington: The Science and Practice of Pharmacy,21st Ed., Lippincott, Williams, and Wilkins, Philadelphia Pa. (2005).

A composition comprising an inhibitor of MAVS and/or agonist of NLRX1can be administered directly to the airways of a subject in the form ofan aerosol or by nebulization. For use as aerosols, an inhibitor of MAVSand/or agonist of NLRX in solution or suspension may be packaged in apressurized aerosol container together with suitable propellants, forexample, hydrocarbon propellants like propane, butane, or isobutane withconventional adjuvants. An inhibitor of MAVS and/or agonist of NLRX1 canalso be administered in a non-pressurized form such as in a nebulizer oratomizer.

The term “nebulization” is well known in the art to include reducingliquid to a fine spray. Preferably, by such nebulization small liquiddroplets of uniform size are produced from a larger body of liquid in acontrolled manner. Nebulization can be achieved by any suitable meanstherefore, including by using many nebulizers known and marketed today.For example, an AEROMIST pneumatic nebulizer available from InhalationPlastic, Inc. of Niles, Ill. When the active ingredients are adapted tobe administered, either together or individually, via nebulizer(s) theycan be in the form of a nebulized aqueous suspension or solution, withor without a suitable pH or tonicity adjustment, either as a unit doseor multidose device.

As is well known, any suitable gas can be used to apply pressure duringthe nebulization, with preferred gases to date being those which arechemically inert to a modulator of an inhibitor of MAVS and/or agonistof NLRX1. Exemplary gases including, but are not limited to, nitrogen,argon or helium can be used to high advantage.

In some embodiments, an inhibitor of MAVS and/or agonist of NLRX1 canalso be administered directly to the airways in the form of a drypowder. For use as a dry powder, an inhibitor of MAVS and/or agonist ofNLRX1 can be administered by use of an inhaler. Exemplary inhalersinclude metered dose inhalers and dry powdered inhalers.

A metered dose inhaler or “MDI” is a pressure resistant canister orcontainer filled with a product such as a pharmaceutical compositiondissolved in a liquefied propellant or micronized particles suspended ina liquefied propellant. The propellants which can be used includechlorofluorocarbons, hydrocarbons or hydrofluoroalkanes. Especiallypreferred propellants are P134a (tetrafluoroethane) and P227(heptafluoropropane) each of which may be used alone or in combination.They are optionally used in combination with one or more otherpropellants and/or one or more surfactants and/or one or more otherexcipients, for example ethanol, a lubricant, an anti-oxidant and/or astabilizing agent. The correct dosage of the composition is delivered tothe patient.

A dry powder inhaler (i.e. Turbuhaler (Astra AB)) is a system operablewith a source of pressurized air to produce dry powder particles of apharmaceutical composition that is compacted into a very small volume.

Dry powder aerosols for inhalation therapy are generally produced withmean diameters primarily in the range of <5 μm. As the diameter ofparticles exceeds 3 μm, there is increasingly less phagocytosis bymacrophages. However, increasing the particle size also has been foundto minimize the probability of particles (possessing standard massdensity) entering the airways and acini due to excessive deposition inthe oropharyngeal or nasal regions.

Suitable powder compositions include, by way of illustration, powderedpreparations of an inhibitor of MAVS and/or agonist of NLRX1 thoroughlyintermixed with lactose, or other inert powders acceptable forintrabronchial administration. The powder compositions can beadministered via an aerosol dispenser or encased in a breakable capsulewhich may be inserted by the patient into a device that punctures thecapsule and blows the powder out in a steady stream suitable forinhalation. The compositions can include propellants, surfactants, andco-solvents and may be filled into conventional aerosol containers thatare closed by a suitable metering valve.

Aerosols for the delivery to the respiratory tract are known in the art.See for example, Adjei, A. and Garren, J. Pharm. Res., 1: 565-569(1990); Zanen, P. and Lamm, J.-W. J. Int. J. Pharm., 114: 111-115(1995); Gonda, I. “Aerosols for delivery of therapeutic an diagnosticagents to the respiratory tract,” in Critical Reviews in TherapeuticDrug Carrier Systems, 6:273-313 (1990); Anderson et al., Am. Rev.Respir. Dis., 140: 1317-1324 (1989)) and have potential for the systemicdelivery of peptides and proteins as well (Patton and Platz, AdvancedDrug Delivery Reviews, 8:179-196 (1992)); Timsina et. al., Int. J.Pharm., 101: 1-13 (1995); and Tansey, I. P., Spray Technol. Market,4:26-29 (1994); French, D. L., Edwards, D. A. and Niven, R. W., AerosolSci., 27: 769-783 (1996); Visser, J., Powder Technology 58: 1-10(1989)); Rudt, S. and R. H. Muller, J. Controlled Release, 22: 263-272(1992); Tabata, Y, and Y. Ikada, Biomed. Mater. Res., 22: 837-858(1988); Wall, D. A., Drug Delivery, 2: 10 1-20 1995); Patton, J. andPlatz, R., Adv. Drug Del. Rev., 8: 179-196 (1992); Bryon, P., Adv. Drug.Del. Rev., 5: 107-132 (1990); Patton, J. S., et al., Controlled Release,28: 15 79-85 (1994); Damms, B. and Bains, W., Nature Biotechnology(1996); Niven, R. W., et al., Pharm. Res., 12(9); 1343-1349 (1995); andKobayashi, S., et al., Pharm. Res., 13(1): 80-83 (1996), contents of allof which are herein incorporated by reference in their entirety.

Conventional dosage forms generally provide rapid or immediate drugrelease from the formulation. Depending on the pharmacology andpharmacokinetics of the drug, use of conventional dosage forms can leadto wide fluctuations in the concentrations of the drug in a patient'sblood and other tissues. These fluctuations can impact a number ofparameters, such as dose frequency, onset of action, duration ofefficacy, maintenance of therapeutic blood levels, toxicity, sideeffects, and the like. Advantageously, controlled-release formulationscan be used to control a drug's onset of action, duration of action,plasma levels within the therapeutic window, and peak blood levels. Inparticular, controlled- or extended-release dosage forms or formulationscan be used to ensure that the maximum effectiveness of a drug isachieved while minimizing potential adverse effects and safety concerns,which can occur both from under-dosing a drug (i.e., going below theminimum therapeutic levels) as well as exceeding the toxicity level forthe drug. In some embodiments, the inhibitor of MAVS and/or agonist ofNLRX1 can be administered in a sustained release formulation.

Controlled-release pharmaceutical products have a common goal ofimproving drug therapy over that achieved by their non-controlledrelease counterparts. Ideally, the use of an optimally designedcontrolled-release preparation in medical treatment is characterized bya minimum of drug substance being employed to cure or control thecondition in a minimum amount of time. Advantages of controlled-releaseformulations include: 1) extended activity of the drug; 2) reduceddosage frequency; 3) increased patient compliance; 4) usage of lesstotal drug; 5) reduction in local or systemic side effects; 6)minimization of drug accumulation; 7) reduction in blood levelfluctuations; 8) improvement in efficacy of treatment; 9) reduction ofpotentiation or loss of drug activity; and 10) improvement in speed ofcontrol of diseases or conditions. Kim, Cherng-ju, Controlled ReleaseDosage Form Design, 2 (Technomic Publishing, Lancaster, Pa.: 2000).

Most controlled-release formulations are designed to initially releasean amount of drug (active ingredient) that promptly produces the desiredtherapeutic effect, and gradually and continually release other amountsof drug to maintain this level of therapeutic or prophylactic effectover an extended period of time. In order to maintain this constantlevel of drug in the body, the drug must be released from the dosageform at a rate that will replace the amount of drug being metabolizedand excreted from the body. Controlled-release of an active ingredientcan be stimulated by various conditions including, but not limited to,pH, ionic strength, osmotic pressure, temperature, enzymes, water, andother physiological conditions or compounds.

A variety of known controlled- or extended-release dosage forms,formulations, and devices can be adapted for use with the salts andcompositions of the disclosure. Examples include, but are not limitedto, those described in U.S. Pat. Nos. 3,845,770; 3,916,899; 3,536,809;3,598,123; 4,008,719; 5,674,533; 5,059,595; 5,591,767; 5,120,548;5,073,543; 5,639,476; 5,354,556; 5,733,566; and 6,365,185 B1; each ofwhich is incorporated herein by reference. These dosage forms can beused to provide slow or controlled-release of one or more activeingredients using, for example, hydroxypropylmethyl cellulose, otherpolymer matrices, gels, permeable membranes, osmotic systems (such asOROS® (Alza Corporation, Mountain View, Calif. USA)), or a combinationthereof to provide the desired release profile in varying proportions.

The methods described herein can further comprise administering a secondagent and/or treatment to the subject, e.g. as part of a combinatorialtherapy. By way of non-limiting example, if a subject is to be treatedwith an NLRX1 agonist according to the methods described herein, thesubject can also be administered a second agent and/or treatment knownto be beneficial for subjects suffering from COPD, emphysema, and/orcigarette smoke-induced lung damage. Examples of such agents and/ortreatments include, but are not limited to, administration of abronchodilator; administration of an inhaled steroid; administration ofoxygen therapy; bullectomy; lung volume reduction surgery; and smokingcessation.

In certain embodiments, an effective dose of a composition comprising aninhibitor of MAVS and/or an agonist of NLRX1 as described herein can beadministered to a patient once. In certain embodiments, an effectivedose of a composition comprising an inhibitor of MAVS and/or an agonistof NLRX1 can be administered to a patient repeatedly. For systemicadministration, subjects can be administered a therapeutic amount of acomposition comprising an inhibitor of MAVS and/or an agonist of NLRX1,such as, e.g. 0.1 mg/kg, 0.5 mg/kg, 1.0 mg/kg, 2.0 mg/kg, 2.5 mg/kg, 5mg/kg, 10 mg/kg, 15 mg/kg, 20 mg/kg, 25 mg/kg, 30 mg/kg, 40 mg/kg, 50mg/kg, or more.

In some embodiments, after an initial treatment regimen, the treatmentscan be administered on a less frequent basis. For example, aftertreatment biweekly for three months, treatment can be repeated once permonth, for six months or a year or longer. Treatment according to themethods described herein can reduce levels of a marker or symptom of acondition, e.g. by at least 10%, at least 15%, at least 20%, at least25%, at least 30%, at least 40%, at least 50%, at least 60%, at least70%, at least 80% or at least 90% or more.

The dosage of a composition as described herein can be determined by aphysician and adjusted, as necessary, to suit observed effects of thetreatment. With respect to duration and frequency of treatment, it istypical for skilled clinicians to monitor subjects in order to determinewhen the treatment is providing therapeutic benefit, and to determinewhether to increase or decrease dosage, increase or decreaseadministration frequency, discontinue treatment, resume treatment, ormake other alterations to the treatment regimen. The dosing schedule canvary from once a week to daily depending on a number of clinicalfactors, such as the subject's sensitivity to the active ingredient. Thedesired dose or amount of activation can be administered at one time ordivided into subdoses, e.g., 2-4 subdoses and administered over a periodof time, e.g., at appropriate intervals through the day or otherappropriate schedule. In some embodiments, administration can bechronic, e.g., one or more doses and/or treatments daily over a periodof weeks or months. Examples of dosing and/or treatment schedules areadministration daily, twice daily, three times daily or four or moretimes daily over a period of 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month,2 months, 3 months, 4 months, 5 months, or 6 months, or more. Acomposition comprising an inhibitor of MAVS and/or an agonist of NLRX1can be administered over a period of time, such as over a 5 minute, 10minute, 15 minute, 20 minute, or 25 minute period.

The dosage ranges for the administration of an inhibitor of MAVS and/oran agonist of NLRX1, according to the methods described herein dependupon, for example, the form of the inhibitor of MAVS and/or agonist ofNLRX1, its potency, and the extent to which symptoms, markers, orindicators of a condition described herein are desired to be reduced,for example the percentage reduction desired for inflammation or theextent to which, for example, NLRX1 expression levels are desired to beinduced. The dosage should not be so large as to cause adverse sideeffects. Generally, the dosage will vary with the age, condition, andsex of the patient and can be determined by one of skill in the art. Thedosage can also be adjusted by the individual physician in the event ofany complication.

The efficacy of an inhibitor of MAVS and/or an agonist of NLRX1 in, e.g.the treatment of a condition described herein, or to induce a responseas described herein (e.g. NLRX1 activity and/or expression) can bedetermined by the skilled clinician. However, a treatment is considered“effective treatment,” as the term is used herein, if one or more of thesigns or symptoms of a condition described herein are altered in abeneficial manner, other clinically accepted symptoms are improved, oreven ameliorated, or a desired response is induced e.g., by at least 10%following treatment according to the methods described herein. Efficacycan be assessed, for example, by measuring a marker, indicator, symptom,and/or the incidence of a condition treated according to the methodsdescribed herein or any other measurable parameter appropriate. Efficacycan also be measured by a failure of an individual to worsen as assessedby hospitalization, or need for medical interventions (i.e., progressionof the disease is halted). Methods of measuring these indicators areknown to those of skill in the art and/or are described herein.Treatment includes any treatment of a disease in an individual or ananimal (some non-limiting examples include a human or an animal) andincludes: (1) inhibiting the disease, e.g., preventing a worsening ofsymptoms (e.g. pain or inflammation); or (2) relieving the severity ofthe disease, e.g., causing regression of symptoms. An effective amountfor the treatment of a disease means that amount which, whenadministered to a subject in need thereof, is sufficient to result ineffective treatment as that term is defined herein, for that disease.Efficacy of an agent can be determined by assessing physical indicatorsof a condition or desired response, (e.g. NLRX1 activity and/orexpression). It is well within the ability of one skilled in the art tomonitor efficacy of administration and/or treatment by measuring any oneof such parameters, or any combination of parameters. Efficacy can beassessed in animal models of a condition described herein, for exampletreatment of emphysema and/or COPD. When using an experimental animalmodel, efficacy of treatment is evidenced when a statisticallysignificant change in a marker is observed, e.g. lung volume, surfacearea, and/or chord length.

In vitro and animal model assays are provided herein which allow theassessment of a given dose of inhibitor of MAVS and/or an agonist ofNLRX1. By way of non-limiting example, the effects of a dose ofinhibitor of MAVS and/or an agonist of NLRX1 can be assessed bymeasuring the levels of expression of CXCL13, MMP-12, cathepsisn K,cathepsin S, type 1 IFNs, caspase 1, IL-1β, and/or IL-18, whereincreased NLRX1 levels and/or activity results in decreased levels ofCXCL13, MMP-12, cathepsisn K, cathepsin S, type 1 IFNs, caspase 1,IL-1β, and/or IL-18 and where decreased MAVS levels and/or activityresults in decreased levels of CXCL13, MMP-12, cathepsisn K, cathepsinS, type 1 IFNs, caspase 1, IL-1β, and/or IL-18.

The efficacy of a given dosage combination can also be assessed in ananimal model, e.g. a mouse model of cigarette smoke-induced lung damage.For example, mice can be exposed to cigarette smoke and a therapeuticagent or control intranasally. Lung tissue can be harvested at thedesired time point and mean chord length and the surface area of thelungs determined following stereological analysis of the lungs accordingto the ATS/ERS guidelines. Briefly, the left lung can be inflated with0.5% low temperature-melting agarose in 10% buffered formalin fixativeat a constant pressure of 25 cm. After the fixation, lung volume (V_(L))of the left lung was determined by the water immersion method. Imagescan be taken equally spaced and systematically placed meander-like overthe whole surface of the lung sections. Pictures can be quantitativelyanalyzed by using a test system of points and lines superimposed overthe digital images via the STEPanizer™ program. The intersecting pointsfalling on alveolar space (Pa), alveolar ducts (Pd), and points fallingon septum (Ps) can be counted separately among total points (Ptotal).Point counts yielded relative volume densities and alveolar surface area(S) can be calculated by the following formula; (1) Airspace fraction(F_(A))=(Pa+Pd+Ps)/Ptotal; (2) Airspace volume (V_(A))=F_(A)×V_(L); (3)S=4V_(A)/Lm (mean linear intercept).

In one aspect, described herein is a kit for performing any of theassays and/or methods described herein. In some embodiments, the kit cancomprise a NLRX1-specific reagent.

A kit is any manufacture (e.g., a package or container) comprising atleast one reagent, e.g., an antibody reagent(s) or nucleic acid probe,for specifically detecting, e.g., a NLRX1 expression product or fragmentthereof, the manufacture being promoted, distributed, or sold as a unitfor performing the methods or assays described herein. When the kits,and methods described herein are used for diagnosis and/or treatment ofCOPD, emphysema, and/or cigarette-induced lung damage in patients, thereagents (e.g., detection probes) or systems can be selected such that apositive result is obtained in at least about 20%, at least about 40%,at least about 60%, at least about 80%, at least about 90%, at leastabout 95%, at least about 99% or in 100% of subjects having ordeveloping COPD, emphysema, and/or cigarette-induced lung damage.

In some embodiments, described herein is a kit for the detection of aNLRX1 expression product in a sample, the kit comprising at least afirst NLRX1-specific reagent as described herein which specificallybinds the NLRX1 expression product, on a solid support and comprising adetectable label. The kits described herein include reagents and/orcomponents that permit assaying the level of an expression product in asample obtained from a subject (e.g., a biological sample obtained froma subject). The kits described herein can optionally comprise additionalcomponents useful for performing the methods and assays describedherein.

A kit can further comprise devices and/or reagents for concentrating anexpression product (e.g, a polypeptide) in a sample, e.g. a plasmasample. Thus, ultrafiltration devices permitting, e.g., proteinconcentration from plasma can also be included as a kit component.

Preferably, a diagnostic or prognostic kit for use with the methods andassays disclosed herein contains detection reagents for NLRX1 expressionproducts. Such detection reagents comprise in addition to NLRX1-specificreagents, for example, buffer solutions, labels or washing liquids etc.Furthermore, the kit can comprise an amount of a known nucleic acidand/or polypeptide, which can be used for a calibration of the kit or asan internal control. A diagnostic kit for the detection of an expressionproduct can also comprise accessory ingredients like secondary affinityligands, e.g., secondary antibodies, detection dyes and any othersuitable compound or liquid necessary for the performance of aexpression product detection method known to the person skilled in theart. Such ingredients are known to the person skilled in the art and mayvary depending on the detection method carried out. Additionally, thekit may comprise an instruction leaflet and/or may provide informationas to the relevance of the obtained results.

In some aspects, the invention described herein is directed to systems(and computer readable media for causing computer systems) for obtainingdata from at least one sample obtained from at least one subject, thesystem comprising 1) a measuring module configured to receive the atleast one sample and perform at least one analysis on the at least onesample to determine the level and/or activity of NLRX1 in the sample; 2)a storage device configured to store data output from the determinationmodule; and 3) a display module for displaying a content based in parton the data output from the determination module, wherein the contentcomprises a signal indicative of the level and/or activity of NRLX1.

In one embodiment, provided herein is a system comprising: (a) at leastone memory containing at least one computer program adapted to controlthe operation of the computer system to implement a method that includesa measuring module configured to measure the level of NLRX1 in a testsample obtained from a subject; a storage module configured to storeoutput data from the determination module; a comparison module adaptedto compare the data stored on the storage module with a reference level,and to provide a retrieved content, and a display module for displayingwhether the sample comprises a level of NRLX1 which is significantlydecreases relative to the reference expression level and/or displayingthe relative level of NRLX1 and (b) at least one processor for executingthe computer program (see FIG. 17).

The term “computer” can refer to any non-human apparatus that is capableof accepting a structured input, processing the structured inputaccording to prescribed rules, and producing results of the processingas output. Examples of a computer include: a computer; a general purposecomputer; a supercomputer; a mainframe; a super mini-computer; amini-computer; a workstation; a micro-computer; a server; an interactivetelevision; a hybrid combination of a computer and an interactivetelevision; a tablet; and application-specific hardware to emulate acomputer and/or software. A computer can have a single processor ormultiple processors, which can operate in parallel and/or not inparallel. A computer also refers to two or more computers connectedtogether via a network for transmitting or receiving information betweenthe computers. An example of such a computer includes a distributedcomputer system for processing information via computers linked by anetwork.

The term “computer-readable medium” may refer to any storage device usedfor storing data accessible by a computer, as well as any other meansfor providing access to data by a computer. Examples of astorage-device-type computer-readable medium include: a magnetic harddisk; a floppy disk; an optical disk, such as a CD-ROM and a DVD; amagnetic tape; a memory chip. The term a “computer system” may refer toa system having a computer, where the computer comprises acomputer-readable medium embodying software to operate the computer. Theterm “software” is used interchangeably herein with “program” and refersto prescribed rules to operate a computer. Examples of software include:software; code segments; instructions; computer programs; and programmedlogic.

The computer readable storage media can be any available tangible mediathat can be accessed by a computer. Computer readable storage mediaincludes volatile and nonvolatile, removable and non-removable tangiblemedia implemented in any method or technology for storage of informationsuch as computer readable instructions, data structures, program modulesor other data. Computer readable storage media includes, but is notlimited to, RAM (random access memory), ROM (read only memory), EPROM(erasable programmable read only memory), EEPROM (electrically erasableprogrammable read only memory), flash memory or other memory technology,CD-ROM (compact disc read only memory), DVDs (digital versatile disks)or other optical storage media, magnetic cassettes, magnetic tape,magnetic disk storage or other magnetic storage media, other types ofvolatile and non-volatile memory, and any other tangible medium whichcan be used to store the desired information and which can accessed by acomputer including and any suitable combination of the foregoing.

Computer-readable data embodied on one or more computer-readable mediamay define instructions, for example, as part of one or more programsthat, as a result of being executed by a computer, instruct the computerto perform one or more of the functions described herein, and/or variousembodiments, variations and combinations thereof. Such instructions maybe written in any of a plurality of programming languages, for example,Java, J#, Visual Basic, C, C#, C++, Fortran, Pascal, Eiffel, Basic,COBOL assembly language, and the like, or any of a variety ofcombinations thereof. The computer-readable media on which suchinstructions are embodied may reside on one or more of the components ofeither of a system, or a computer readable storage medium describedherein, may be distributed across one or more of such components.

The computer-readable media may be transportable such that theinstructions stored thereon can be loaded onto any computer resource toimplement the aspects of the present invention discussed herein. Inaddition, it should be appreciated that the instructions stored on thecomputer-readable medium, described above, are not limited toinstructions embodied as part of an application program running on ahost computer. Rather, the instructions may be embodied as any type ofcomputer code (e.g., software or microcode) that can be employed toprogram a computer to implement aspects of the present invention. Thecomputer executable instructions may be written in a suitable computerlanguage or combination of several languages. Basic computationalbiology methods are known to those of ordinary skill in the art and aredescribed in, for example, Setubal and Meidanis et al., Introduction toComputational Biology Methods (PWS Publishing Company, Boston, 1997);Salzberg, Searles, Kasif, (Ed.), Computational Methods in MolecularBiology, (Elsevier, Amsterdam, 1998); Rashidi and Buehler,Bioinformatics Basics: Application in Biological Science and Medicine(CRC Press, London, 2000) and Ouelette and Bzevanis Bioinformatics: APractical Guide for Analysis of Gene and Proteins (Wiley & Sons, Inc.,2nd ed., 2001).

Embodiments of the invention can be described through functionalmodules, which are defined by computer executable instructions recordedon computer readable media and which cause a computer to perform methodsteps when executed. The modules are segregated by function for the sakeof clarity. However, it should be understood that the modules/systemsneed not correspond to discreet blocks of code and the describedfunctions can be carried out by the execution of various code portionsstored on various media and executed at various times. Furthermore, itshould be appreciated that the modules can perform other functions, thusthe modules are not limited to having any particular functions or set offunctions.

The functional modules of certain embodiments of the invention includeat minimum a measuring module, a storage module, a computing module, anda display module. The functional modules can be executed on one, ormultiple, computers, or by using one, or multiple, computer networks.The measuring module has computer executable instructions to providee.g., levels of expression products etc in computer readable form.

The measuring module can comprise any system for detecting a signalelicited from an assay to determine the level and/or activity of NLRX1described above herein. In some embodiments, such systems can include aninstrument, e.g., AU2700 (Beckman Coulter Brea, Calif.) as describedherein for quantitative measurement of polypeptides or e.g., a real timePCR machine, e.g. a LIGHTCYCLER™ (Roche). In some embodiments, themeasuring module can measure the intensity of a detectable signal froman assay indicating the level of NLRX1 polypeptide in the test sample.In some embodiments, the assay can be an immunoassay. In someembodiments, the measuring module can measure the intensity of adetectable signal from a RT-PCR assay indicating the level of NLRX1 RNAtranscript in the test sample.

The information determined in the determination system can be read bythe storage module. As used herein the “storage module” is intended toinclude any suitable computing or processing apparatus or other deviceconfigured or adapted for storing data or information. Examples ofelectronic apparatus suitable for use with the present invention includestand-alone computing apparatus, data telecommunications networks,including local area networks (LAN), wide area networks (WAN), Internet,Intranet, and Extranet, and local and distributed computer processingsystems. Storage modules also include, but are not limited to: magneticstorage media, such as floppy discs, hard disc storage media, magnetictape, optical storage media such as CD-ROM, DVD, electronic storagemedia such as RAM, ROM, EPROM, EEPROM and the like, general hard disksand hybrids of these categories such as magnetic/optical storage media.The storage module is adapted or configured for having recorded thereon,for example, sample name, biomolecule assayed and the level of saidbiomolecule. Such information may be provided in digital form that canbe transmitted and read electronically, e.g., via the Internet, ondiskette, via USB (universal serial bus) or via any other suitable modeof communication.

As used herein, “stored” refers to a process for encoding information onthe storage module. Those skilled in the art can readily adopt any ofthe presently known methods for recording information on known media togenerate manufactures comprising expression level information.

In some embodiments of any of the systems described herein, the storagemodule stores the output data from the determination module. Inadditional embodiments, the storage module stores reference informationsuch as levels of NLRX1 in healthy subjects and/or a population ofhealthy subjects.

The “computing module” can use a variety of available software programsand formats for computing the level of NLRX1. Such algorithms are wellestablished in the art. A skilled artisan is readily able to determinethe appropriate algorithms based on the size and quality of the sampleand type of data. The data analysis tools and equations described hereincan be implemented in the computing module of the invention. In oneembodiment, the computing module further comprises a comparison module,which compares the level of NLRX1 in a sample obtained from a subject asdescribed herein with the mean value of NLRX1 in a population of healthysubjects (FIG. 18). By way of an example, when the value of NLRX1 in asample obtained from a subject is measured, a comparison module cancompare or match the output data with the mean value of NLRX1 in apopulation of healthy subjects. In certain embodiments, the mean valueof NLRX1 in a population of healthy subjects can be pre-stored in thestorage module. In various embodiments, the comparison module can beconfigured using existing commercially-available or freely-availablesoftware for comparison purpose, and may be optimized for particulardata comparisons that are conducted.

The computing and/or comparison module, or any other module of theinvention, can include an operating system (e.g., UNIX) on which runs arelational database management system, a World Wide Web application, anda World Wide Web server. World Wide Web application includes theexecutable code necessary for generation of database language statements(e.g., Structured Query Language (SQL) statements). Generally, theexecutables will include embedded SQL statements. In addition, the WorldWide Web application may include a configuration file which containspointers and addresses to the various software entities that comprisethe server as well as the various external and internal databases whichmust be accessed to service user requests. The Configuration file alsodirects requests for server resources to the appropriate hardware—as maybe necessary should the server be distributed over two or more separatecomputers. In one embodiment, the World Wide Web server supports aTCP/IP protocol. Local networks such as this are sometimes referred toas “Intranets.” An advantage of such Intranets is that they allow easycommunication with public domain databases residing on the World WideWeb (e.g., the GenBank or Swiss Pro World Wide Web site). In someembodiments users can directly access data (via Hypertext links forexample) residing on Internet databases using a HTML interface providedby Web browsers and Web servers (FIG. 19).

The computing and/or comparison module provides a computer readablecomparison result that can be processed in computer readable form bypredefined criteria, or criteria defined by a user, to provide contentbased in part on the comparison result that may be stored and output asrequested by a user using an output module, e.g., a display module.

In some embodiments, the content displayed on the display module can bethe level of NLRX1 in the sample obtained from a subject. In someembodiments, the content displayed on the display module can be therelative level of NLRX1 in the sample obtained from a subject ascompared to the mean level of NRLX1 in a population of healthy subjects.In some embodiments, if the computing module determines that the levelof NRLX1 in the test sample obtained from a subject is less by astatistically significant amount than the reference level, the displaymodule displays a signal indicating that the levels in the sampleobtained from a subject are less than those of the reference level. Insome embodiments, the signal indicates the subject is in need oftreatment for COPD, emphysema, and/or cigarette smoke-induced lungdamage. In some embodiments, the signal indicates the degree to whichthe level of NRLX1 in the sample obtained from a subject varies from thereference level. In some embodiments, the content displayed on thedisplay module can indicate whether the subject has an increasedlikelihood of having or developing COPD, emphysema, and/or cigarettesmoke-induced lung damage. In some embodiments, the content displayed onthe display module can be a numerical value indicating one of theserisks or probabilities. In such embodiments, the probability can beexpressed in percentages or a fraction. For example, higher percentageor a fraction closer to 1 indicates a higher likelihood of a subjecthaving or developing COPD, emphysema, and/or cigarette smoke-inducedlung damage. In some embodiments, the content displayed on the displaymodule can be single word or phrases to qualitatively indicate a risk orprobability. For example, a word “unlikely” can be used to indicate alower risk for having or developing COPD, emphysema, and/or cigarettesmoke-induced lung damage, while “likely” can be used to indicate a highrisk for having or developing COPD, emphysema, and/or cigarettesmoke-induced lung damage.

In one embodiment of the invention, the content based on the computingand/or comparison result is displayed on a computer monitor. In oneembodiment of the invention, the content based on the computing and/orcomparison result is displayed through printable media. The displaymodule can be any suitable device configured to receive from a computerand display computer readable information to a user. Non-limitingexamples include, for example, general-purpose computers such as thosebased on Intel PENTIUM-type processor, Motorola PowerPC, Sun UltraSPARC,Hewlett-Packard PA-RISC processors, any of a variety of processorsavailable from Advanced Micro Devices (AMD) of Sunnyvale, Calif., or anyother type of processor, visual display devices such as flat paneldisplays, cathode ray tubes and the like, as well as computer printersof various types.

In one embodiment, a World Wide Web browser is used for providing a userinterface for display of the content based on the computing/comparisonresult. It should be understood that other modules of the invention canbe adapted to have a web browser interface. Through the Web browser, auser can construct requests for retrieving data from thecomputing/comparison module. Thus, the user will typically point andclick to user interface elements such as buttons, pull down menus,scroll bars and the like conventionally employed in graphical userinterfaces.

Systems and computer readable media described herein are merelyillustrative embodiments of the invention for determining the leveland/or activity of NLRX1 in a sample obtained from a subject, andtherefore are not intended to limit the scope of the invention.Variations of the systems and computer readable media described hereinare possible and are intended to fall within the scope of the invention.

The modules of the machine, or those used in the computer readablemedium, may assume numerous configurations. For example, function may beprovided on a single machine or distributed over multiple machines.

For convenience, the meaning of some terms and phrases used in thespecification, examples, and appended claims, are provided below. Unlessstated otherwise, or implicit from context, the following terms andphrases include the meanings provided below. The definitions areprovided to aid in describing particular embodiments, and are notintended to limit the claimed invention, because the scope of theinvention is limited only by the claims. Unless otherwise defined, alltechnical and scientific terms used herein have the same meaning ascommonly understood by one of ordinary skill in the art to which thisinvention belongs. If there is an apparent discrepancy between the usageof a term in the art and its definition provided herein, the definitionprovided within the specification shall prevail.

For convenience, certain terms employed herein, in the specification,examples and appended claims are collected here.

The terms “decrease”, “reduced”, “reduction”, or “inhibit” are all usedherein to mean a decrease by a statistically significant amount. In someembodiments, “reduce,” “reduction” or “decrease” or “inhibit” typicallymeans a decrease by at least 10% as compared to a reference level (e.g.the absence of a given treatment) and can include, for example, adecrease by at least about 10%, at least about 20%, at least about 25%,at least about 30%, at least about 35%, at least about 40%, at leastabout 45%, at least about 50%, at least about 55%, at least about 60%,at least about 65%, at least about 70%, at least about 75%, at leastabout 80%, at least about 85%, at least about 90%, at least about 95%,at least about 98%, at least about 99%, or more. As used herein,“reduction” or “inhibition” does not encompass a complete inhibition orreduction as compared to a reference level. “Complete inhibition” is a100% inhibition as compared to a reference level. A decrease can bepreferably down to a level accepted as within the range of normal for anindividual without a given disorder.

The terms “increased”, “increase”, “enhance”, or “activate” are all usedherein to mean an increase by a statically significant amount. In someembodiments, the terms “increased”, “increase”, “enhance”, or “activate”can mean an increase of at least 10% as compared to a reference level,for example an increase of at least about 20%, or at least about 30%, orat least about 40%, or at least about 50%, or at least about 60%, or atleast about 70%, or at least about 80%, or at least about 90% or up toand including a 100% increase or any increase between 10-100% ascompared to a reference level, or at least about a 2-fold, or at leastabout a 3-fold, or at least about a 4-fold, or at least about a 5-foldor at least about a 10-fold increase, or any increase between 2-fold and10-fold or greater as compared to a reference level. In the context of amarker or symptom, a “increase” is a statistically significant increasein such level.

As used herein, a “subject” means a human or animal. Usually the animalis a vertebrate such as a primate, rodent, domestic animal or gameanimal. Primates include chimpanzees, cynomologous monkeys, spidermonkeys, and macaques, e.g., Rhesus. Rodents include mice, rats,woodchucks, ferrets, rabbits and hamsters. Domestic and game animalsinclude cows, horses, pigs, deer, bison, buffalo, feline species, e.g.,domestic cat, canine species, e.g., dog, fox, wolf, avian species, e.g.,chicken, emu, ostrich, and fish, e.g., trout, catfish and salmon. Insome embodiments, the subject is a mammal, e.g., a primate, e.g., ahuman. The terms, “individual,” “patient” and “subject” are usedinterchangeably herein.

Preferably, the subject is a mammal. The mammal can be a human,non-human primate, mouse, rat, dog, cat, horse, or cow, but is notlimited to these examples. Mammals other than humans can beadvantageously used as subjects that represent animal models of COPD,emphysema, and/or cigarette smoke-induced lung damage. A subject can bemale or female.

As used herein, “engineered” refers to the aspect of having beenmanipulated by the hand of man. For example, a NLRX1 polypeptide isconsidered to be “engineered” when the sequence of the polypeptideand/or encoding nucleic acid sequence manipulated by the hand of man todiffer from the sequence of an polypeptide as it exists in nature. As iscommon practice and is understood by those in the art, progeny andcopies of an engineered polynucleotide and/or polypeptide are typicallystill referred to as “engineered” even though the actual manipulationwas performed on a prior entity.

As used herein, “recombinant” refers to a cell, tissue or organism thathas undergone transformation with a new combination of genes or DNA.When used in reference to nucleic acid molecules, “recombinant” refersto a combination of nucleic acid molecules that are joined togetherusing recombinant DNA technology into a progeny nucleic acid molecule,and/or a heterologous nucleic acid sequence introduced into a cell,tissue, or organism. When used in reference to a polypeptide,“recombinant” refers to a polypeptide which is the expression product ofa recombinant nucleic acid, and can be such a polypeptide as produced bya recombinant cell, tissue, or organism. The nucleic acid molecule canbe stably integrated into the genome of the host or the nucleic acidmolecule can also be present as an extrachromosomal molecule. Such anextrachromosomal molecule can be auto-replicating. Recombinant viruses,cells, and organisms are understood to encompass not only the endproduct of a transformation process, but also recombinant progenythereof.

A subject can be one who has been previously diagnosed with oridentified as suffering from or having a condition in need of treatment(e.g. COPD, emphysema, and/or cigarette smoke-induced lung damage) orone or more complications related to such a condition, and optionally,have already undergone treatment for the condition or the one or morecomplications related to the condition. Alternatively, a subject canalso be one who has not been previously diagnosed as having a conditionor one or more complications related to the condition. For example, asubject can be one who exhibits one or more risk factors for a conditionor one or more complications related to the condition or a subject whodoes not exhibit risk factors.

A “subject in need” of treatment for a particular condition can be asubject having that condition, diagnosed as having that condition, or atrisk of developing that condition.

As used herein, the terms “protein” and “polypeptide” are usedinterchangeably herein to designate a series of amino acid residues,connected to each other by peptide bonds between the alpha-amino andcarboxy groups of adjacent residues. The terms “protein”, and“polypeptide” refer to a polymer of amino acids, including modifiedamino acids (e.g., phosphorylated, glycated, glycosylated, etc.) andamino acid analogs, regardless of its size or function. “Protein” and“polypeptide” are often used in reference to relatively largepolypeptides, whereas the term “peptide” is often used in reference tosmall polypeptides, but usage of these terms in the art overlaps. Theterms “protein” and “polypeptide” are used interchangeably herein whenreferring to a gene product and fragments thereof. Thus, exemplarypolypeptides or proteins include gene products, naturally occurringproteins, homologs, orthologs, paralogs, fragments and otherequivalents, variants, fragments, and analogs of the foregoing.

As used herein, a particular “polypeptide”, e.g. a NLRX1 polypeptide caninclude the human polypeptide (e.g., SEQ ID NO: 2); as well as homologsfrom other species, including but not limited to bovine, dog, catchicken, murine, rat, porcine, ovine, turkey, horse, fish, baboon andother primates. The terms also refer to fragments or variants of thenative polypeptide that maintain at least 50% of the activity or effectof the native full length polypeptide, e.g. as measured in anappropriate animal model. Conservative substitution variants thatmaintain the activity of wildtype polypeptides will include aconservative substitution as defined herein. The identification of aminoacids most likely to be tolerant of conservative substitution whilemaintaining at least 50% of the activity of the wildtype is guided by,for example, sequence alignment with homologs or paralogs from otherspecies. Amino acids that are identical between homologs are less likelyto tolerate change, while those showing conservative differences areobviously much more likely to tolerate conservative change in thecontext of an artificial variant. Similarly, positions withnon-conservative differences are less likely to be critical to functionand more likely to tolerate conservative substitution in an artificialvariant. Variants can be tested for activity, for example, byadministering the variant to an appropriate animal model of allograftrejection as described herein.

In some embodiments, a polypeptide, e.g., a NLRx1 polypeptide, can be avariant of a sequence described herein, e.g. a variant of a NLRX1polypeptide comprising the amino acid sequence of SEQ ID NO: 2. In someembodiments, the variant is a conservative substitution variant.Variants can be obtained by mutations of native nucleotide sequences,for example. A “variant,” as referred to herein, is a polypeptidesubstantially homologous to a native or reference polypeptide, but whichhas an amino acid sequence different from that of the native orreference polypeptide because of one or a plurality of deletions,insertions or substitutions. Polypeptide-encoding DNA sequencesencompass sequences that comprise one or more additions, deletions, orsubstitutions of nucleotides when compared to a native or reference DNAsequence, but that encode a variant protein or fragment thereof thatretains the relevant biological activity relative to the referenceprotein, e.g., at least 50% of the wildtype reference protein. As toamino acid sequences, one of skill will recognize that individualsubstitutions, deletions or additions to a nucleic acid, peptide,polypeptide, or protein sequence which alters a single amino acid or asmall percentage, (i.e. 5% or fewer, e.g. 4% or fewer, or 3% or fewer,or 1% or fewer) of amino acids in the encoded sequence is a“conservatively modified variant” where the alteration results in thesubstitution of an amino acid with a chemically similar amino acid. Itis contemplated that some changes can potentially improve the relevantactivity, such that a variant, whether conservative or note, has morethan 100% of the activity of wildtype, e.g. 110%, 125%, 150%, 175%,200%, 500%, 1000% or more.

One method of identifying amino acid residues which can be substitutedis to align, for example, the human polypeptide to a homolog from one ormore non-human species. Alignment can provide guidance regarding notonly residues likely to be necessary for function but also, conversely,those residues likely to tolerate change. Where, for example, analignment shows two identical or similar amino acids at correspondingpositions, it is more likely that that site is important functionally.Where, conversely, alignment shows residues in corresponding positionsto differ significantly in size, charge, hydrophobicity, etc., it ismore likely that that site can tolerate variation in a functionalpolypeptide. The variant amino acid or DNA sequence can be at least 90%,at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, ormore, identical to a native or reference sequence, e.g. SEQ ID NOs: 2 or4 or a nucleic acid encoding one of those amino acid sequences. Thedegree of homology (percent identity) between a native and a mutantsequence can be determined, for example, by comparing the two sequencesusing freely available computer programs commonly employed for thispurpose on the world wide web. The variant amino acid or DNA sequencecan be at least 90%, at least 91%, at least 92%, at least 93%, at least94%, at least 95%, at least 96%, at least 97%, at least 98%, at least99%, or more, similar to the sequence from which it is derived (referredto herein as an “original” sequence). The degree of similarity (percentsimilarity) between an original and a mutant sequence can be determined,for example, by using a similarity matrix. Similarity matrices are wellknown in the art and a number of tools for comparing two sequences usingsimilarity matrices are freely available online, e.g. BLASTp (availableon the world wide web at http://blast.ncbi.nlm.nih.gov), with defaultparameters set.

A given amino acid can be replaced by a residue having similarphysiochemical characteristics, e.g., substituting one aliphatic residuefor another (such as Ile, Val, Leu, or Ala for one another), orsubstitution of one polar residue for another (such as between Lys andArg; Glu and Asp; or Gln and Asn). Other such conservativesubstitutions, e.g., substitutions of entire regions having similarhydrophobicity characteristics, are well known. Polypeptides comprisingconservative amino acid substitutions can be tested in any one of theassays described herein to confirm that a desired activity of a nativeor reference polypeptide is retained. Conservative substitution tablesproviding functionally similar amino acids are well known in the art.Such conservatively modified variants are in addition to and do notexclude polymorphic variants, interspecies homologs, and allelesconsistent with the disclosure. Typically conservative substitutions forone another include: 1) Alanine (A), Glycine (G); 2) Aspartic acid (D),Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R),Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V);6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W); 7) Serine (S),Threonine (T); and 8) Cysteine (C), Methionine (M) (see, e.g.,Creighton, Proteins (1984)).

Any cysteine residue not involved in maintaining the proper conformationof the polypeptide also can be substituted, generally with serine, toimprove the oxidative stability of the molecule and prevent aberrantcrosslinking. Conversely, cysteine bond(s) can be added to thepolypeptide to improve its stability or facilitate oligomerization.

In some embodiments, a polypeptide, e.g., a NLRX1 polypeptide,administered to a subject can comprise one or more amino acidsubstitutions or modifications. In some embodiments, the substitutionsand/or modifications can prevent or reduce proteolytic degradationand/or prolong half-life of the polypeptide in the subject. In someembodiments, a polypeptide can be modified by conjugating or fusing itto other polypeptide or polypeptide domains such as, by way ofnon-limiting example, transferrin (WO06096515A2), albumin (Yeh et al.,1992), growth hormone (US2003104578AA); cellulose (Levy and Shoseyov,2002); and/or Fc fragments (Ashkenazi and Chamow, 1997). The referencesin the foregoing paragraph are incorporated by reference herein in theirentireties.

In some embodiments, a polypeptide, e.g., an NLRX1 polypeptide, asdescribed herein can comprise at least one peptide bond replacement. Asingle peptide bond or multiple peptide bonds, e.g. 2 bonds, 3 bonds, 4bonds, 5 bonds, or 6 or more bonds, or all the peptide bonds can bereplaced. An isolated peptide as described herein can comprise one typeof peptide bond replacement or multiple types of peptide bondreplacements, e.g. 2 types, 3 types, 4 types, 5 types, or more types ofpeptide bond replacements. Non-limiting examples of peptide bondreplacements include urea, thiourea, carbamate, sulfonyl urea,trifluoroethylamine, ortho-(aminoalkyl)-phenylacetic acid,para-(aminoalkyl)-phenylacetic acid, meta-(aminoalkyl)-phenylaceticacid, thioamide, tetrazole, boronic ester, olefinic group, andderivatives thereof.

In some embodiments, a polypeptide, e.g., a NLRX1 polypeptide, asdescribed herein can comprise naturally occurring amino acids commonlyfound in polypeptides and/or proteins produced by living organisms, e.g.Ala (A), Val (V), Leu (L), Ile (I), Pro (P), Phe (F), Trp (W), Met (M),Gly (G), Ser (S), Thr (T), Cys (C), Tyr (Y), Asn (N), Gln (Q), Asp (D),Glu (E), Lys (K), Arg (R), and His (H). In some embodiments, an NLRX1polypeptide as described herein can comprise alternative amino acids.Non-limiting examples of alternative amino acids include, D-amino acids;beta-amino acids; homocysteine, phosphoserine, phosphothreonine,phosphotyrosine, hydroxyproline, gamma-carboxyglutamate; hippuric acid,octahydroindole-2-carboxylic acid, statine,1,2,3,4,-tetrahydroisoquinoline-3-carboxylic acid, penicillamine(3-mercapto-D-valine), ornithine, citruline, alpha-methyl-alanine,para-benzoylphenylalanine, para-amino phenylalanine,p-fluorophenylalanine, phenylglycine, propargylglycine, sarcosine, andtert-butylglycine), diaminobutyric acid,7-hydroxy-tetrahydroisoquinoline carboxylic acid, naphthylalanine,biphenylalanine, cyclohexylalanine, amino-isobutyric acid, norvaline,norleucine, tert-leucine, tetrahydroisoquinoline carboxylic acid,pipecolic acid, phenylglycine, homophenylalanine, cyclohexylglycine,dehydroleucine, 2,2-diethylglycine, 1-amino-1-cyclopentanecarboxylicacid, 1-amino-1-cyclohexanecarboxylic acid, amino-benzoic acid,amino-naphthoic acid, gamma-aminobutyric acid, difluorophenylalanine,nipecotic acid, alpha-amino butyric acid, thienyl-alanine,t-butylglycine, trifluorovaline; hexafluoroleucine; fluorinated analogs;azide-modified amino acids; alkyne-modified amino acids; cyano-modifiedamino acids; and derivatives thereof.

In some embodiments, a polypeptide, e.g. a NLRX1 polypeptide, can bemodified, e.g. by addition of a moiety to one or more of the amino acidscomprising the peptide. In some embodiments, a polypeptide as describedherein can comprise one or more moiety molecules, e.g. 1 or more moietymolecules per peptide, 2 or more moiety molecules per peptide, 5 or moremoiety molecules per peptide, 10 or more moiety molecules per peptide ormore moiety molecules per peptide. In some embodiments, a polypeptide asdescribed herein can comprise one more types of modifications and/ormoieties, e.g. 1 type of modification, 2 types of modifications, 3 typesof modifications or more types of modifications. Non-limiting examplesof modifications and/or moieties include PEGylation; glycosylation;HESylation; ELPylation; lipidation; acetylation; amidation; end-cappingmodifications; cyano groups; phosphorylation; albumin, and cyclization.In some embodiments, an end-capping modification can compriseacetylation at the N-terminus, N-terminal acylation, and N-terminalformylation. In some embodiments, an end-capping modification cancomprise amidation at the C-terminus, introduction of C-terminalalcohol, aldehyde, ester, and thioester moieties. The half-life of apolypeptide can be increased by the addition of moieties, e.g. PEG oralbumin.

In some embodiments, the polypeptide administered to the subject (or anucleic acid encoding such a polypeptide) can be a functional fragmentof one of the amino acid sequences described herein. As used herein, a“functional fragment” is a fragment or segment of a peptide whichretains at least 50% of the wildtype reference polypeptide's activityaccording to the assays described below herein. A functional fragmentcan comprise conservative substitutions of the sequences disclosedherein.

Alterations of the original amino acid sequence can be accomplished byany of a number of techniques known to one of skill in the art.Mutations can be introduced, for example, at particular loci bysynthesizing oligonucleotides containing a mutant sequence, flanked byrestriction sites permitting ligation to fragments of the nativesequence. Following ligation, the resulting reconstructed sequenceencodes an analog having the desired amino acid insertion, substitution,or deletion. Alternatively, oligonucleotide-directed site-specificmutagenesis procedures can be employed to provide an altered nucleotidesequence having particular codons altered according to the substitution,deletion, or insertion required. Techniques for making such alterationsinclude those disclosed by Walder et al. (Gene 42:133, 1986); Bauer etal. (Gene 37:73, 1985); Craik (BioTechniques, January 1985, 12-19);Smith et al. (Genetic Engineering: Principles and Methods, Plenum Press,1981); and U.S. Pat. Nos. 4,518,584 and 4,737,462, which are hereinincorporated by reference in their entireties. In some embodiments, apolypeptide as described herein can be chemically synthesized andmutations can be incorporated as part of the chemical synthesis process.

In some embodiments, a polypeptide, e.g., a NLRX1 polypeptide, asdescribed herein can be formulated as a pharmaceutically acceptableprodrug. As used herein, a “prodrug” refers to compounds that can beconverted via some chemical or physiological process (e.g., enzymaticprocesses and metabolic hydrolysis) to a therapeutic agent. Thus, theterm “prodrug” also refers to a precursor of a biologically activecompound that is pharmaceutically acceptable. A prodrug may be inactivewhen administered to a subject, i.e. an ester, but is converted in vivoto an active compound, for example, by hydrolysis to the free carboxylicacid or free hydroxyl. The prodrug compound often offers advantages ofsolubility, tissue compatibility or delayed release in an organism. Theterm “prodrug” is also meant to include any covalently bonded carriers,which release the active compound in vivo when such prodrug isadministered to a subject. Prodrugs of an active compound may beprepared by modifying functional groups present in the active compoundin such a way that the modifications are cleaved, either in routinemanipulation or in vivo, to the parent active compound. Prodrugs includecompounds wherein a hydroxy, amino or mercapto group is bonded to anygroup that, when the prodrug of the active compound is administered to asubject, cleaves to form a free hydroxy, free amino or free mercaptogroup, respectively. Examples of prodrugs include, but are not limitedto, acetate, formate and benzoate derivatives of an alcohol oracetamide, formamide and benzamide derivatives of an amine functionalgroup in the active compound and the like. See Harper, “DrugLatentiation” in Jucker, ed. Progress in Drug Research 4:221-294 (1962);Morozowich et al, “Application of Physical Organic Principles to ProdrugDesign” in E. B. Roche ed. Design of Biopharmaceutical Propertiesthrough Prodrugs and Analogs, APHA Acad. Pharm. Sci. 40 (1977);Bioreversible Carriers in Drug in Drug Design, Theory and Application,E. B. Roche, ed., APHA Acad. Pharm. Sci. (1987); Design of Prodrugs, H.Bundgaard, Elsevier (1985); Wang et al. “Prodrug approaches to theimproved delivery of peptide drug” in Curr. Pharm. Design. 5(4):265-287(1999); Pauletti et al. (1997) Improvement in peptide bioavailability:Peptidomimetics and Prodrug Strategies, Adv. Drug. Delivery Rev.27:235-256; Mizen et al. (1998) “The Use of Esters as Prodrugs for OralDelivery of (3-Lactam antibiotics,” Pharm. Biotech. 11:345-365;Gaignault et al. (1996) “Designing Prodrugs and Bioprecursors I. CarrierProdrugs,” Pract. Med. Chem. 671-696; Asgharnej ad, “Improving Oral DrugTransport”, in Transport Processes in Pharmaceutical Systems, G. L.Amidon, P. I. Lee and E. M. Topp, Eds., Marcell Dekker, p. 185-218(2000); Balant et al., “Prodrugs for the improvement of drug absorptionvia different routes of administration”, Eur. J. Drug Metab.Pharmacokinet., 15(2): 143-53 (1990); Balimane and Sinko, “Involvementof multiple transporters in the oral absorption of nucleosideanalogues”, Adv. Drug Delivery Rev., 39(1-3): 183-209 (1999); Browne,“Fosphenytoin (Cerebyx)”, Clin. Neuropharmacol. 20(1): 1-12 (1997);Bundgaard, “Bioreversible derivatization of drugs—principle andapplicability to improve the therapeutic effects of drugs”, Arch. Pharm.Chemi 86(1): 1-39 (1979); Bundgaard H. “Improved drug delivery by theprodrug approach”, Controlled Drug Delivery 17: 179-96 (1987); BundgaardH. “Prodrugs as a means to improve the delivery of peptide drugs”, Arfv.Drug Delivery Rev. 8(1): 1-38 (1992); Fleisher et al. “Improved oraldrug delivery: solubility limitations overcome by the use of prodrugs”,Arfv. Drug Delivery Rev. 19(2): 115-130 (1996); Fleisher et al. “Designof prodrugs for improved gastrointestinal absorption by intestinalenzyme targeting”, Methods Enzymol. 112 (Drug Enzyme Targeting, Pt. A):360-81, (1985); Farquhar D, et al., “Biologically ReversiblePhosphate-Protective Groups”, Pharm. Sci., 72(3): 324-325 (1983);Freeman S, et al., “Bioreversible Protection for the Phospho Group:Chemical Stability and Bioactivation of Di(4-acetoxy-benzyl)Methylphosphonate with Carboxyesterase,” Chem. Soc., Chem. Commun.,875-877 (1991); Friis and Bundgaard, “Prodrugs of phosphates andphosphonates: Novel lipophilic alphaacyloxyalkyl ester derivatives ofphosphate- or phosphonate containing drugs masking the negative chargesof these groups”, Eur. J. Pharm. Sci. 4: 49-59 (1996); Gangwar et al.,“Prodrug, molecular structure and percutaneous delivery”, Des. Biopharm.Prop. Prodrugs Analogs, [Symp.] Meeting Date 1976, 409-21. (1977);Nathwani and Wood, “Penicillins: a current review of their clinicalpharmacology and therapeutic use”, Drugs 45(6): 866-94 (1993); Sinhababuand Thakker, “Prodrugs of anticancer agents”, Adv. Drug Delivery Rev.19(2): 241-273 (1996); Stella et al., “Prodrugs. Do they have advantagesin clinical practice?”, Drugs 29(5): 455-73 (1985); Tan et al.“Development and optimization of anti-HIV nucleoside analogs andprodrugs: A review of their cellular pharmacology, structure-activityrelationships and pharmacokinetics”, Adv. Drug Delivery Rev. 39(1-3):117-151 (1999); Taylor, “Improved passive oral drug delivery viaprodrugs”, Adv. Drug Delivery Rev., 19(2): 131-148 (1996); Valentino andBorchardt, “Prodrug strategies to enhance the intestinal absorption ofpeptides”, Drug Discovery Today 2(4): 148-155 (1997); Wiebe and Knaus,“Concepts for the design of anti-HIV nucleoside prodrugs for treatingcephalic HIV infection”, Adv. Drug Delivery Rev.: 39(1-3):63-80 (1999);Waller et al., “Prodrugs”, Br. J. Clin. Pharmac. 28: 497-507 (1989),which are incorporated by reference herein in their entireties.

In some embodiments, a polypeptide as described herein can be apharmaceutically acceptable solvate. The term “solvate” refers to apeptide as described herein in the solid state, wherein molecules of asuitable solvent are incorporated in the crystal lattice. A suitablesolvent for therapeutic administration is physiologically tolerable atthe dosage administered. Examples of suitable solvents for therapeuticadministration are ethanol and water. When water is the solvent, thesolvate is referred to as a hydrate. In general, solvates are formed bydissolving the compound in the appropriate solvent and isolating thesolvate by cooling or using an antisolvent. The solvate is typicallydried or azeotroped under ambient conditions.

The peptides of the present invention can be synthesized by using wellknown methods including recombinant methods and chemical synthesis.Recombinant methods of producing a peptide through the introduction of avector including nucleic acid encoding the peptide into a suitable hostcell is well known in the art, such as is described in Sambrook et al.,Molecular Cloning: A Laboratory Manual, 2d Ed, Vols 1 to 8, Cold SpringHarbor, N.Y. (1989); M. W. Pennington and B. M. Dunn, Methods inMolecular Biology: Peptide Synthesis Protocols, Vol 35, Humana Press,Totawa, N.J. (1994), contents of both of which are herein incorporatedby reference. Peptides can also be chemically synthesized using methodswell known in the art. See for example, Merrifield et al., J. Am. Chem.Soc. 85:2149 (1964); Bodanszky, M., Principles of Peptide Synthesis,Springer-Verlag, New York, N.Y. (1984); Kimmerlin, T. and Seebach, D. J.Pept. Res. 65:229-260 (2005); Nilsson et al., Annu. Rev. Biophys.Biomol. Struct. (2005) 34:91-118; W. C. Chan and P. D. White (Eds.) FmocSolid Phase Peptide Synthesis: A Practical Approach, Oxford UniversityPress, Cary, N.C. (2000); N. L. Benoiton, Chemistry of PeptideSynthesis, CRC Press, Boca Raton, Fla. (2005); J. Jones, Amino Acid andPeptide Synthesis, 2^(nd) Ed, Oxford University Press, Cary, N.C.(2002); and P. Lloyd-Williams, F. Albericio, and E. Giralt, ChemicalApproaches to the synthesis of peptides and proteins, CRC Press, BocaRaton, Fla. (1997), contents of all of which are herein incorporated byreference. Peptide derivatives can also be prepared as described in U.S.Pat. Nos. 4,612,302; 4,853,371; and 4,684,620, and U.S. Pat. App. Pub.No. 2009/0263843, contents of all which are herein incorporated byreference.

In some embodiments, the technology described herein relates to anucleic acid encoding a polypeptide (e.g. a NLRX1 polypeptide) asdescribed herein. As used herein, the term “nucleic acid” or “nucleicacid sequence” refers to any molecule, preferably a polymeric molecule,incorporating units of ribonucleic acid, deoxyribonucleic acid or ananalog thereof. The nucleic acid can be either single-stranded ordouble-stranded. A single-stranded nucleic acid can be one strandnucleic acid of a denatured double-stranded DNA. Alternatively, it canbe a single-stranded nucleic acid not derived from any double-strandedDNA. In one aspect, the nucleic acid is DNA. In another aspect, thenucleic acid is RNA. Suitable nucleic acid molecules are DNA, includinggenomic DNA or cDNA. Other suitable nucleic acid molecules are RNA,including mRNA. The nucleic acid molecule can be naturally occurring, asin genomic DNA, or it may be synthetic, i.e., prepared based up humanaction, or may be a combination of the two. The nucleic acid moleculecan also have certain modification such as 2′-deoxy, 2′-deoxy-2′-fluoro,2′-O-methyl, 2′-O-methoxyethyl (2′-O-MOE), 2′-O-aminopropyl (2′-O-AP),2′-O-dimethylaminoethyl (2′-O-DMAOE), 2′-O-dimethylaminopropyl(2′-O-DMAP), 2′-O-dimethylaminoethyloxyethyl (2′-O-DMAEOE), or2′-O—N-methylacetamido (2′-O-NMA), cholesterol addition, andphosphorothioate backbone as described in US Patent Application20070213292; and certain ribonucleoside that are is linked between the2′-oxygen and the 4′-carbon atoms with a methylene unit as described inU.S. Pat. No. 6,268,490, wherein both patent and patent application areincorporated hereby reference in their entirety.

In some embodiments, a nucleic acid encoding a polypeptide as describedherein (e.g. an NLRX1 polypeptide) is comprised by a vector. In some ofthe aspects described herein, a nucleic acid sequence encoding a givenpolypeptide as described herein, or any module thereof, is operablylinked to a vector. The term “vector”, as used herein, refers to anucleic acid construct designed for delivery to a host cell or fortransfer between different host cells. As used herein, a vector can beviral or non-viral. The term “vector” encompasses any genetic elementthat is capable of replication when associated with the proper controlelements and that can transfer gene sequences to cells. A vector caninclude, but is not limited to, a cloning vector, an expression vector,a plasmid, phage, transposon, cosmid, chromosome, virus, virion, etc.

As used herein, the term “expression vector” refers to a vector thatdirects expression of an RNA or polypeptide from sequences linked totranscriptional regulatory sequences on the vector. The sequencesexpressed will often, but not necessarily, be heterologous to the cell.An expression vector may comprise additional elements, for example, theexpression vector may have two replication systems, thus allowing it tobe maintained in two organisms, for example in human cells forexpression and in a prokaryotic host for cloning and amplification. Theterm “expression” refers to the cellular processes involved in producingRNA and proteins and as appropriate, secreting proteins, including whereapplicable, but not limited to, for example, transcription, transcriptprocessing, translation and protein folding, modification andprocessing. “Expression products” include RNA transcribed from a gene,and polypeptides obtained by translation of mRNA transcribed from agene. The term “gene” means the nucleic acid sequence which istranscribed (DNA) to RNA in vitro or in vivo when operably linked toappropriate regulatory sequences. The gene may or may not includeregions preceding and following the coding region, e.g. 5′ untranslated(5′UTR) or “leader” sequences and 3′ UTR or “trailer” sequences, as wellas intervening sequences (introns) between individual coding segments(exons).

As used herein, the term “viral vector” refers to a nucleic acid vectorconstruct that includes at least one element of viral origin and has thecapacity to be packaged into a viral vector particle. The viral vectorcan contain the nucleic acid encoding a polypeptide as described hereinin place of non-essential viral genes. The vector and/or particle may beutilized for the purpose of transferring any nucleic acids into cellseither in vitro or in vivo. Numerous forms of viral vectors are known inthe art.

By “recombinant vector” is meant a vector that includes a heterologousnucleic acid sequence, or “transgene” that is capable of expression invivo. It should be understood that the vectors described herein can, insome embodiments, be combined with other suitable compositions andtherapies. In some embodiments, the vector is episomal. The use of asuitable episomal vector provides a means of maintaining the nucleotideof interest in the subject in high copy number extra chromosomal DNAthereby eliminating potential effects of chromosomal integration.

In some embodiments, an inhibitor of a given polypeptide can be anantibody reagent specific for that polypeptide. As used herein an“antibody” refers to IgG, IgM, IgA, IgD or IgE molecules orantigen-specific antibody fragments thereof (including, but not limitedto, a Fab, F(ab′)₂, Fv, disulphide linked Fv, scFv, single domainantibody, closed conformation multispecific antibody, disulphide-linkedscfv, diabody), whether derived from any species that naturally producesan antibody, or created by recombinant DNA technology; whether isolatedfrom serum, B-cells, hybridomas, transfectomas, yeast or bacteria.

As described herein, an “antigen” is a molecule that is bound by abinding site on an antibody agent. Typically, antigens are bound byantibody ligands and are capable of raising an antibody response invivo. An antigen can be a polypeptide, protein, nucleic acid or othermolecule or portion thereof. The term “antigenic determinant” refers toan epitope on the antigen recognized by an antigen-binding molecule, andmore particularly, by the antigen-binding site of said molecule.

As used herein, the term “antibody reagent” refers to a polypeptide thatincludes at least one immunoglobulin variable domain or immunoglobulinvariable domain sequence and which specifically binds a given antigen.An antibody reagent can comprise an antibody or a polypeptide comprisingan antigen-binding domain of an antibody. In some embodiments, anantibody reagent can comprise a monoclonal antibody or a polypeptidecomprising an antigen-binding domain of a monoclonal antibody. Forexample, an antibody can include a heavy (H) chain variable region(abbreviated herein as VH), and a light (L) chain variable region(abbreviated herein as VL). In another example, an antibody includes twoheavy (H) chain variable regions and two light (L) chain variableregions. The term “antibody reagent” encompasses antigen-bindingfragments of antibodies (e.g., single chain antibodies, Fab and sFabfragments, F(ab′)2, Fd fragments, Fv fragments, scFv, and domainantibodies (dAb) fragments (see, e.g. de Wildt et al., Eur J. Immunol.1996; 26(3):629-39; which is incorporated by reference herein in itsentirety)) as well as complete antibodies. An antibody can have thestructural features of IgA, IgG, IgE, IgD, IgM (as well as subtypes andcombinations thereof). Antibodies can be from any source, includingmouse, rabbit, pig, rat, and primate (human and non-human primate) andprimatized antibodies. Antibodies also include midibodies, humanizedantibodies, chimeric antibodies, and the like.

The VH and VL regions can be further subdivided into regions ofhypervariability, termed “complementarity determining regions” (“CDR”),interspersed with regions that are more conserved, termed “frameworkregions” (“FR”). The extent of the framework region and CDRs has beenprecisely defined (see, Kabat, E. A., et al. (1991) Sequences ofProteins of Immunological Interest, Fifth Edition, U.S. Department ofHealth and Human Services, NIH Publication No. 91-3242, and Chothia, C.et al. (1987) J. Mol. Biol. 196:901-917; which are incorporated byreference herein in their entireties). Each VH and VL is typicallycomposed of three CDRs and four FRs, arranged from amino-terminus tocarboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3,CDR3, FR4.

The terms “antigen-binding fragment” or “antigen-binding domain”, whichare used interchangeably herein are used to refer to one or morefragments of a full length antibody that retain the ability tospecifically bind to a target of interest. Examples of binding fragmentsencompassed within the term “antigen-binding fragment” of a full lengthantibody include (i) a Fab fragment, a monovalent fragment consisting ofthe VL, VH, CL and CH1 domains; (ii) a F(ab′)2 fragment, a bivalentfragment including two Fab fragments linked by a disulfide bridge at thehinge region; (iii) an Fd fragment consisting of the VH and CH1 domains;(iv) an Fv fragment consisting of the VL and VH domains of a single armof an antibody, (v) a dAb fragment (Ward et al., (1989) Nature341:544-546; which is incorporated by reference herein in its entirety),which consists of a VH or VL domain; and (vi) an isolatedcomplementarity determining region (CDR) that retains specificantigen-binding functionality.

As used herein, the term “specific binding” refers to a chemicalinteraction between two molecules, compounds, cells and/or particleswherein the first entity binds to the second, target entity with greaterspecificity and affinity than it binds to a third entity which is anon-target. In some embodiments, specific binding can refer to anaffinity of the first entity for the second target entity which is atleast 10 times, at least 50 times, at least 100 times, at least 500times, at least 1000 times or greater than the affinity for the thirdnontarget entity. A reagent specific for a given target is one thatexhibits specific binding for that target under the conditions of theassay being utilized.

Additionally, and as described herein, a recombinant humanized antibodycan be further optimized to decrease potential immunogenicity, whilemaintaining functional activity, for therapy in humans. In this regard,functional activity means a polypeptide capable of displaying one ormore known functional activities associated with a recombinant antibodyor antibody reagent thereof as described herein. Such functionalactivities include, e.g. the ability to bind to e.g., NLRX1 or MAVS.

As used herein, “expression level” refers to the number of mRNAmolecules and/or polypeptide molecules encoded by a given gene that arepresent in a cell or sample. Expression levels can be increased ordecreased relative to a reference level.

As used herein, the terms “treat,” “treatment,” “treating,” or“amelioration” refer to therapeutic treatments, wherein the object is toreverse, alleviate, ameliorate, inhibit, slow down or stop theprogression or severity of a condition associated with a disease ordisorder, e.g. COPD, emphysema, and/or cigarette smoke-induced lungdamage. The term “treating” includes reducing or alleviating at leastone adverse effect or symptom of a condition, disease or disorderassociated with a condition. Treatment is generally “effective” if oneor more symptoms or clinical markers are reduced. Alternatively,treatment is “effective” if the progression of a disease is reduced orhalted. That is, “treatment” includes not just the improvement ofsymptoms or markers, but also a cessation of, or at least slowing of,progress or worsening of symptoms compared to what would be expected inthe absence of treatment. Beneficial or desired clinical resultsinclude, but are not limited to, alleviation of one or more symptom(s),diminishment of extent of disease, stabilized (i.e., not worsening)state of disease, delay or slowing of disease progression, ameliorationor palliation of the disease state, remission (whether partial ortotal), and/or decreased mortality, whether detectable or undetectable.The term “treatment” of a disease also includes providing relief fromthe symptoms or side-effects of the disease (including palliativetreatment).

As used herein, the term “pharmaceutical composition” refers to theactive agent in combination with a pharmaceutically acceptable carriere.g. a carrier commonly used in the pharmaceutical industry. The phrase“pharmaceutically acceptable” is employed herein to refer to thosecompounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of human beings and animals without excessive toxicity,irritation, allergic response, or other problem or complication,commensurate with a reasonable benefit/risk ratio.

As used herein, the term “administering,” refers to the placement of acompound as disclosed herein into a subject by a method or route whichresults in at least partial delivery of the agent at a desired site.Pharmaceutical compositions comprising the compounds disclosed hereincan be administered by any appropriate route which results in aneffective treatment in the subject.

The term “statistically significant” or “significantly” refers tostatistical significance and generally means a two standard deviation(2SD) or greater difference.

Other than in the operating examples, or where otherwise indicated, allnumbers expressing quantities of ingredients or reaction conditions usedherein should be understood as modified in all instances by the term“about.” The term “about” when used in connection with percentages canmean±1%.

As used herein the term “comprising” or “comprises” is used in referenceto compositions, methods, and respective component(s) thereof, that areessential to the method or composition, yet open to the inclusion ofunspecified elements, whether essential or not.

The term “consisting of” refers to compositions, methods, and respectivecomponents thereof as described herein, which are exclusive of anyelement not recited in that description of the embodiment.

As used herein the term “consisting essentially of” refers to thoseelements required for a given embodiment. The term permits the presenceof elements that do not materially affect the basic and novel orfunctional characteristic(s) of that embodiment.

The singular terms “a,” “an,” and “the” include plural referents unlesscontext clearly indicates otherwise. Similarly, the word “or” isintended to include “and” unless the context clearly indicatesotherwise. Although methods and materials similar or equivalent to thosedescribed herein can be used in the practice or testing of thisdisclosure, suitable methods and materials are described below. Theabbreviation, “e.g.” is derived from the Latin exempli gratia, and isused herein to indicate a non-limiting example. Thus, the abbreviation“e.g.” is synonymous with the term “for example.”

Definitions of common terms in cell biology and molecular biology can befound in “The Merck Manual of Diagnosis and Therapy”, 19th Edition,published by Merck Research Laboratories, 2006 (ISBN 0-911910-19-0);Robert S. Porter et al. (eds.), The Encyclopedia of Molecular Biology,published by Blackwell Science Ltd., 1994 (ISBN 0-632-02182-9); TheELISA guidebook (Methods in molecular biology 149) by Crowther J. R.(2000); Fundamentals of RIA and Other Ligand Assays by Jeffrey Travis,1979, Scientific Newsletters; Immunology by Werner Luttmann, publishedby Elsevier, 2006. Definitions of common terms in molecular biology canalso be found in Benjamin Lewin, Genes X, published by Jones & BartlettPublishing, 2009 (ISBN-10: 0763766321); Kendrew et al. (eds.), MolecularBiology and Biotechnology: a Comprehensive Desk Reference, published byVCH Publishers, Inc., 1995 (ISBN 1-56081-569-8) and Current Protocols inProtein Sciences 2009, Wiley Intersciences, Coligan et al., eds.

Unless otherwise stated, the present invention was performed usingstandard procedures, as described, for example in Sambrook et al.,Molecular Cloning: A Laboratory Manual (4 ed.), Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., USA (2012); CurrentProtocols in Protein Science (CPPS) (John E. Coligan, et. al., ed., JohnWiley and Sons, Inc.), Current Protocols in Cell Biology (CPCB) (Juan S.Bonifacino et. al. ed., John Wiley and Sons, Inc.), and Culture ofAnimal Cells: A Manual of Basic Technique by R. Ian Freshney, Publisher:Wiley-Liss; 5th edition (2005), which are all incorporated by referenceherein in their entireties.

Other terms are defined herein within the description of the variousaspects of the invention.

All patents and other publications; including literature references,issued patents, published patent applications, and co-pending patentapplications; cited throughout this application are expresslyincorporated herein by reference for the purpose of describing anddisclosing, for example, the methodologies described in suchpublications that might be used in connection with the technologydescribed herein. These publications are provided solely for theirdisclosure prior to the filing date of the present application. Nothingin this regard should be construed as an admission that the inventorsare not entitled to antedate such disclosure by virtue of priorinvention or for any other reason. All statements as to the date orrepresentation as to the contents of these documents is based on theinformation available to the applicants and does not constitute anyadmission as to the correctness of the dates or contents of thesedocuments.

The description of embodiments of the disclosure is not intended to beexhaustive or to limit the disclosure to the precise form disclosed.While specific embodiments of, and examples for, the disclosure aredescribed herein for illustrative purposes, various equivalentmodifications are possible within the scope of the disclosure, as thoseskilled in the relevant art will recognize. For example, while methodsteps or functions are presented in a given order, alternativeembodiments may perform functions in a different order, or functions maybe performed substantially concurrently. The teachings of the disclosureprovided herein can be applied to other procedures or methods asappropriate. The various embodiments described herein can be combined toprovide further embodiments. Aspects of the disclosure can be modified,if necessary, to employ the compositions, functions and concepts of theabove references and application to provide yet further embodiments ofthe disclosure. Moreover, due to biological functional equivalencyconsiderations, some changes can be made in protein structure withoutaffecting the biological or chemical action in kind or amount. These andother changes can be made to the disclosure in light of the detaileddescription. All such modifications are intended to be included withinthe scope of the appended claims.

Specific elements of any of the foregoing embodiments can be combined orsubstituted for elements in other embodiments. Furthermore, whileadvantages associated with certain embodiments of the disclosure havebeen described in the context of these embodiments, other embodimentsmay also exhibit such advantages, and not all embodiments neednecessarily exhibit such advantages to fall within the scope of thedisclosure.

The technology described herein is further illustrated by the followingexamples which in no way should be construed as being further limiting.

Some embodiments of the technology described herein can be definedaccording to any of the following numbered paragraphs:

-   -   1. A method of treating COPD in a subject in need thereof, the        method comprising administering an inhibitor of MAVS.    -   2. A method of treating cigarette smoke-induced lung damage in a        subject in need thereof, the method comprising administering an        inhibitor of MAVS.    -   3. The method of paragraph 2, wherein the cigarette        smoke-induced lung damage is selected from the group consisting        of:        -   inflammation; alveolar destruction; protease induction;            structural cell apoptosis; and inflammasome activation.    -   4. A method of treating emphysema in a subject in need thereof,        the method comprising administering an inhibitor of MAVS.    -   5. The method of paragraph 4, wherein the emphysema is cigarette        smoke induced emphysema.    -   6. The method of any of paragraphs 1-5, wherein the inhibitor of        MAVS is an agonist of NLRX1.    -   7. The method of any of paragraphs 1-5, wherein the inhibitor of        MAVS is a modulator of NLRX1-dependent pathways.    -   8. The method of paragraph 6, wherein the agonist of NLRX1 is a        nucleic acid encoding NLRX1 or an NLRX1 polypeptide.    -   9. A method of treating COPD in a subject in need thereof, the        method comprising administering an agonist of NLRX1.    -   10. A method of treating cigarette smoke-induced lung damage in        a subject in need thereof, the method comprising administering        an agonist of NLRX1.    -   11. The method of paragraph 10, wherein the cigarette        smoke-induced lung damage is selected from the group consisting        of:        -   inflammation; alveolar destruction; protease induction;            structural cell apoptosis; and inflammasome activation.    -   12. A method of treating emphysema in a subject in need thereof,        the method comprising administering an agonist of NLRX1.    -   13. The method of paragraph 12, wherein the emphysema is        cigarette smoke induced emphysema.    -   14. The method of any of paragraphs 1-13, wherein the subject is        a subject determined to have a decreased level of NLRX1        expression.    -   15. An assay comprising:        -   measuring the level of NLRX1 in a test sample obtained from            a subject;        -   wherein an decrease in the expression level relative to a            reference level indicates the subject has a higher risk of            having or developing COPD, emphysema, and/or            cigarette-induced lung damage.    -   16. A method of identifying a subject in need of treatment for        COPD, emphysema, and/or cigarette-induced lung damage, the        method comprising:        -   measuring the level of NLRX1 in a test sample obtained from            a subject; and identifying the subject as being in need of            treatment for COPD, emphysema, and/or cigarette-induced lung            damage when the expression level of NLRX1 is decreased            relative to a reference level.    -   17. A method of determining if a subject is at risk for COPD,        emphysema, and/or cigarette-induced lung damage, the method        comprising:        -   measuring the level of NLRX1 in a test sample obtained from            a subject; comparing the level of NLRX1 in the sample to a            reference level of NLRX1; determining that the subject is at            risk for COPD, emphysema, and/or cigarette-induced lung            damage when the level of NLRX1 is decreased relative to a            reference level; and        -   determining that the subject is not at risk for COPD,            emphysema, and/or cigarette-induced lung damage when the            level of NLRX1 is not decreased relative to a reference            level.    -   18. A method of determining the efficacy of a treatment for        COPD, emphysema, and/or cigarette-induced lung damage, the        method comprising:        -   (a) measuring the level of NLRX1 in a test sample obtained            from a subject before administration of the treatment;        -   (b) measuring the level of NLRX1 in a test sample obtained            from a subject after administration of the treatment;        -   (c) determining that the treatment is efficacious when the            expression level determined in step (b) is not decreased            relative to the expression level determined in step (a); and        -   (d) determining that the treatment is not efficacious when            the expression level determined in step (b) is decreased            relative to the expression level determined in step (a).    -   19. A method of treatment for COPD, emphysema, and/or        cigarette-induced lung damage comprising;        -   measuring the level of NLRX1 in a test sample obtained from            a subject;        -   treating the subject when the level of NLRX1 is decreased            relative to a reference level.    -   20. A method of treatment for COPD, emphysema, and/or        cigarette-induced lung damage comprising;        -   treating a subject determined to have a level of NLRX1 which            is decreased relative to a reference level.    -   21. The method of any of paragraphs 19-20, wherein the treatment        comprises a treatment selected from the group consisting of:        -   administration of a bronchodilator; administration of an            inhaled steroid; administration of oxygen therapy;            bullectomy; lung volume reduction surgery; smoking            cessation; lung transplant; administration of an inhibitor            of MAVS; administration of an agonist of NLRX1;            administration of a nucleic acid encoding NLRX1; and            administration of a NLRX1 polypeptide.    -   22. The assay/method of any of paragraphs 15-21, wherein the        level of NLRX1 is determined by measuring the level of a nucleic        acid.    -   23. The assay/method of paragraph 22, wherein the level of NLRX1        is determined by measuring the level of NLRX1 RNA transcript.    -   24. The assay/method of any of paragraphs 22-23, wherein the        level of the nucleic acid is determined using a method selected        from the group consisting of:        -   RT-PCR; quantitative RT-PCR; Northern blot; microarray based            expression analysis; next-generation sequencing; and RNA in            situ hybridization.    -   25. The assay/method of any of paragraphs 15-24, wherein the        level of NLRX1 is determined by measuring the level of NLRX1        polypeptide.    -   26. The assay/method of paragraph 25, wherein the level of the        polypeptide is determined using a method selected from the group        consisting of:        -   Western blot; immunoprecipitation; enzyme-linked            immunosorbent assay (ELISA); radioimmunological assay (RIA);            sandwich assay; fluorescence in situ hybridization (FISH);            immunohistological staining; radioimmunometric assay;            immunofluoresence assay; mass spectroscopy; FACS; and            immunoelectrophoresis assay.    -   27. The assay/method of any of paragraphs 25-26, wherein the        polypeptide level is measured using immunochemistry.    -   28. The assay of paragraph 27, wherein the antibody reagent is        detectably labeled or generates a detectable signal.    -   29. The assay/method of any of paragraphs 15-28, wherein the        expression level of NLRX1 is normalized relative to the        expression level of one or more reference genes or reference        proteins.    -   30. The assay/method of any of paragraphs 15-29, wherein the        reference level of NLRX1 is the expression level of NLRX1 in a        prior sample obtained from the subject.    -   31. The assay/method of any of paragraphs 15-30, wherein the        subject is a subject with a history of smoking.    -   32. The assay/method of any of paragraphs 15-31, wherein the        sample is selected from the group consisting of:        -   a lung biopsy; bronchoalveolar lavage (BAL); sputum; induced            sputum; blood; plasma; and serum.    -   33. The assay/method of any of paragraphs 15-32, further        comprising the step of treating the subject with a treatment        selected from the group consisting of:        -   administration of a bronchodilator; administration of an            inhaled steroid; administration of oxygen therapy;            bullectomy; lung volume reduction surgery; smoking            cessation; lung transplant; administration of an inhibitor            of MAVS; administration of an agonist of NLRX1;            administration of a nucleic acid encoding NLRX1; and            administration of a NLRX1 polypeptide.    -   34. A kit for performing the method/assay of any of paragraphs        15-33.    -   35. The use of an inhibitor of MAVS for treating COPD,        emphysema, or cigarette-induced lung damage in a subject in need        thereof, the use comprising administering an inhibitor of MAVS        to the subject.    -   36. The use of paragraph 35, wherein the cigarette smoke-induced        lung damage is selected from the group consisting of:        -   inflammation; alveolar destruction; protease induction;            structural cell apoptosis; and inflammasome activation.    -   37. The use of paragraph 35, wherein the emphysema is cigarette        smoke induced emphysema.    -   38. The use of any of paragraphs 35-37, wherein the inhibitor of        MAVS is an agonist of NLRX1.    -   39. The use of any of paragraphs 35-38, wherein the inhibitor of        MAVS is a modulator of NLRX1-dependent pathways.    -   40. The method of paragraph 38, wherein the agonist of NLRX1 is        a nucleic acid encoding NLRX1 or an NLRX1 polypeptide.    -   41. The use of an inhibitor of MAVS for treating COPD,        emphysema, or cigarette-induced lung damage in a subject in need        thereof, the use comprising administering an agonist of NLRX1 to        the subject.    -   42. The use of paragraph 41, wherein the cigarette smoke-induced        lung damage is selected from the group consisting of:        -   inflammation; alveolar destruction; protease induction;            structural cell apoptosis; and inflammasome activation.    -   43. The use of paragraph 41, wherein the emphysema is cigarette        smoke induced emphysema.    -   44. The method of any of paragraphs 41-43, wherein the agonist        of NLRX1 is a nucleic acid encoding NLRX1 or an NLRX1        polypeptide.

EXAMPLES Example 1 Suppression of NLRX1 in Chronic Obstructive PulmonaryDisease

It is demonstrated herein that NLRX1 expression is significantlydecreased in three COPD cohorts. This suppression correlated withdisease severity and inversely with pulmonary function, quality of lifeand prognosis. CS inhibited murine NLRX1 and null mutations of NLRX1augmented CS-induced inflammation, alveolar destruction, proteaseinduction, structural cell apoptosis and inflammasome activation. Incontrast, null mutations of MAVS abrogated this CS-induced inflammationand remodeling. Furthermore, restoration of NLRX1 ameliorated CS-inducedalveolar destruction significantly. Thus, CS inhibits NLRX1 whichfacilitates CS-induced and MAVS-dependent inflammatory, remodeling,protease, cell death and inflammasome responses.

Cigarette smoke (CS) causes a broad spectrum of diseases characterizedby inflammation and tissue remodeling including chronic obstructivepulmonary disease (COPD), cancer and atherosclerosis (1-3). In many ofthese disorders the inflammation is believed to drive diseasepathogenesis. This can be seen in, COPD, the fourth leading cause ofdeath in the world, where pulmonary inflammation is believed to becausally related to the emphysema and other pathologic alterations inthe lungs from these patients (3-5).

CS regulates the inflammasome where it activates caspase 1 and IL-18(6,7). IL-18 signaling also plays a key role in the pathogenesis ofCS-induced inflammation and emphysema (6). However, the mechanism(s)that controls lung inflammasome activation at baseline and thealterations that CS induces to activate the inflammasome have not beendefined.

Mitochondria are cellular powerhouses that generate ATP and participatein other critical responses including calcium homeostasis, cellsignaling, apoptosis, and aging (8). Recent studies also demonstratedthat mitochondria play a critical role in cellular innate antiviralimmunity (reviewed in references 9, 10). Viral infections are well knownto play important roles in the pathogenesis of CS-induced disorders suchas COPD (11,12). CS and viruses/viral PAMPs interact to induceexaggerated pulmonary inflammatory and remodeling responses and thatMAVS (mitochondrial antiviral signaling molecule) plays a critical rolein this interaction (5). MAVS is exquisitely regulated by stimulatorssuch as the RIG-like helicases that induce MAVS self-association andinduce inflammation. Importantly, it is now known that MAVS is tonicallyinhibited in the quiescent non-infected state (reviewed in reference13). In addition, the mitochondrial protein, nucleotide-binding,leucine-rich repeats (NLR) molecule X1 (NLRX1) has been reported toinhibit virus activation of MAVS (14-16).

It is hypothesized herein that mitochondria play a critical role in thepathogenesis of CS-induced responses in COPD. Specifically it washypothesized that the tissue effects of CS are mediated by its abilityto abrogate the effects of specific MAVS inhibitors and that NLRX1 is acritical target of this CS-induced dysinhibition. To test thishypothesis the expression of NLRX1 was evaluated in three cohorts ofpatients with COPD and the relationships between NLRX1 suppression anddisease severity were characterized. In addition, the effects of CS onthe expression of NLRX1 were evaluated and the roles of MAVS and NLRX1in CS-induced murine pulmonary inflammation and alveolar remodelingresponses were defined.

RESULTS AND DISCUSSION

First, mRNAs were extracted from fresh frozen lung tissues from the 7controls and 36 patients with COPD in the NIH-sponsored Lung TissueResearch Consortium (LTRC) cohort. The latter contained 6, 10, 10 and 10specimens from GOLD stage 1, 2, 3 and 4 individuals, respectively. Thebasic demographic characteristics of these patients are summarized inthe Table 1. Interestingly, real-time RT-PCR analysis demonstrated thatthe levels of mRNA encoding NLRX1 were significantly decreased inpatients with advanced stages of COPD (FIG. 1A-1B). In keeping with thisobservation, western blot evaluations of mitochondria-enriched proteinfractions demonstrated that the expression of NLRX1 protein wassignificantly decreased in patients with advanced stages of COPD (FIG.1C). In addition, NLRX1 expression correlated significantly with thepatient's forced expiratory volume in one second (FEV₁; % predicted), anindicator of airflow limitation and a measure of COPD disease severity(FIG. 1D-1E). Interestingly, similar correlations were noted betweenNLRX1 suppression and pre- and post-bronchodilator FEV₁ suggesting thatthese relationships are not related to reversible airways obstruction(FIG. 1D-1E). In addition, similar suppression of NLRX1 was seen in acohort of patients from the University of Pittsburgh which was mostimpressive in patients with severe emphysema (FIG. 1F). Because the LTRCand Pittsburgh cohorts did not contain large numbers of patients withmild-moderate disease a third cohort of Korean patients withmild-moderate disease (Asan cohort) was also studied (Table 1C). Thestudies of this Asan cohort demonstrated that, the expression of NLRX1was also significantly decreased in patients with GOLD Stage 1 and 2COPD where it correlated significantly with the patient's FEV₁ (%,predicted) (FIGS. 1G, 1H, 4A, 4B and 5). Almost all human samples werefrom former or current smokers in the three human cohorts (Table 1),preventing the evaluation of smoking effects on NLRX1 expression inthese cohorts. There was no statistical difference of NLRX1 expressionbetween former smokers vs. current smokers in all three human cohorts(FIG. 6 and data not shown). Finally, in all cases, these alterationswere at least partially NLRX1-specific because the expression of relatedgenes including RIG-I, MDA-5, MAVS, NLRP3 and caspases-1, -4 and -5 werenot similarly altered (FIGS. 7A-7B and data not shown). These studiesdemonstrate that the levels of NLRX1 are selectively decreased inpatients with COPD where this suppression correlates with clinical stageand parameters of disease severity.

To further understand the clinical implications of these findings, therelationships between NLRX1 gene expression and clinical parameters ofCOPD were evaluated. These studies demonstrated that, in the LTRCcohort, the levels of NLRX1 gene expression also correlated with othermeasures of pulmonary function including diffusing capacity (DL_(CO))and 6 minute-walking distance (FIGS. 8A-8C and data not shown).Importantly, the levels of NLRX1 mRNA also correlated inversely with theBODE index and scores on the St. George's Respiratory Questionnaire(SGRQ) (FIG. 8B and data not shown) which are predictors of diseasemortality and quality of life respectively. The relationships betweenNLRX1 and dyspnea were also assessed using the BORG scale at rest and atthe termination of exercise. Although the levels of NLRX1 did notcorrelate with the BORG scale at rest (data not shown) they didcorrelate with the BORG scale at exercise termination (FIG. 8C).Importantly, CXCL13, which is produced by lymphoid follicles in COPD(17) and inhibited via an NLRX1-dependent mechanism (see below) wassignificantly enhanced in patients with advanced stages of COPD where itcorrelated inversely with the expression of NLRX1 and also correlatedwith the patient's FEV₁ (%, predicted) and other clinical variablesincluding DL_(CO), the BODE index and SGRQ scores. (FIGS. 9 and 10A-10Band data not shown). These studies reinforce the relationships betweenthe suppression of NLRX1 regulated pathways and abnormal pulmonaryfunction and highlight the relationships between NLRX1 suppression andmortality, poor quality of life and exercise-induced dyspnea.

To define the role(s) of NLRX1 in CS-induced responses, the effects ofCS on NLRX1 expression in WT mice were characterized and the CS-inducedtissue responses in WT and NLRX1 null mutant (−/−) animals were definedusing previously described methodology (5, 6). NLRX1 mRNA and proteinwere readily appreciated in lungs and enriched mitochondria from WT mice(FIG. 2A-2B). Importantly the levels of pulmonary NLRX1 mRNA and NLRX1protein were significantly decreased after CS exposure (FIG. 2A-2B).

Immunohistochemical staining of NLRX1 protein revealed that NLRX1expression was prominent in alveolar macrophages both in mice and humans(data not shown). Western blot analysis of NLRX1 protein in BAL cellsfrom 3 month-CS-exposed lungs where alveolar macrophages consist of morethan 95% of total BAL cells further confirmed that NLRX1 protein wassignificantly suppressed after CS exposure (FIGS. 2C and 11A). Theexpression of MAVS protein, however, was not significantly altered inBAL cells after CS exposure (FIGS. 2C and 11B). This suppression ofNLRX1 was, at least partially, reversible because, NLRX1 gene expressionreturned to normal levels 3 months after the cessation of CS exposure(FIG. 12). In keeping with reported experiments using WT mice, modestBAL and alveolar inflammation and modest alveolar destruction could beappreciated after 3 and 6 months of CS-exposure respectively (6, 18).Importantly, these inflammatory and remodeling responses were markedlyenhanced in lungs from CS-exposed NLRX1^(−/−) mice which manifestenhanced BAL total cell, macrophage and neutrophil recovery (FIG. 2D anddata not shown) and exaggerated emphysema that could be seen withhistologic and morphometric assessments after only 3 months of CSexposure (FIGS. 2E and 2F and data not shown).

To further define the mechanisms by which NLRX1 suppression mediatesCS-induced pulmonary inflammation and alveolar remodeling responses, themolecules/pathways that are believed to play important roles in the COPDpathogenesis were evaluated. In accord with the importance of proteaseanti-protease balance, cell death, viral infection and inflammasomeactivation in COPD pathogenesis (reviewed in references (3, 19-21)), CSinduction of MMP-12 (FIG. 2G), cathepsins K and S (data not shown), type1 IFNs (FIG. 2H), epithelial DNA injury and cell death (FIG. 2I) andcaspase 1, IL-1β and IL-18 activation (FIG. 2J-2L) were enhanced inNLRX1 null mice compared to those from WT mice. Interestingly, CSexposure caused a modest increase in CXCL13 in the lungs of WT mice andthis gene expression was synergistically upregulated in the lungs ofNLRX1 deficient mice after 3 months of CS exposure (FIG. 13). Finally,studies were undertaken to determine if the NLRX1 supplementation couldabrogate the phenotype in the CS exposed mice in vivo. In theseexperiments, a lentiviral gene delivery system was used to expose micethat had been given NLRX1 or controls to CS for 6 months. Theseexperiments demonstrated that CS-induced emphysematous alveolardestruction was markedly decreased in lungs from NLRX1-overexpressedmice compared to those of controls (FIGS. 2M and 14). Taken together,these results indicate that NLRX1 is a critical inhibitor of CS-inducedpulmonary inflammation and tissue remodeling responses, whose inhibitoryfunctions are decreased in pulmonary tissues by chronic CS exposure.

The studies noted above demonstrate that CS exposure decreases NLRX1expression and NLRX1 has been reported to be an inhibitor of MAVS (14,15). Thus studies were next undertaken to determine if mitochondria, inparticular MAVS, plays an important effector role in CS-inducedinflammation and remodeling. First, this was done by comparing theCS-induced responses in WT and MAVS^(−/−) mice. These studiesdemonstrated that inflammation and alveolar destruction weresignificantly decreased in lungs from CS-exposed MAVS^(−/−) micecompared to WT controls (FIG. 3A-3C). CS-induced protease responses,activation of caspase 1, IL-1β and IL-18, TUNEL staining and type 1 IFNproduction were also significantly diminished in the absence of MAVS(FIG. 3D-3G and data not shown). In contrast, CS-induced suppression ofNLRX1 was not altered in lungs from MAVS^(−/−) mice exposed to CS for upto 6 months (FIG. 3H). Next, to determine if MAVS played a criticalroles in the enhancement CS-induced inflammation and alveolardestruction observed in NLRX1^(−/−) mice, NLRX1 and MAVS double mutant(NLRX1^(−/−)/MAVS^(−/−)) mice and appropriate controls were exposed toCS for 3 months. These studies demonstrated that the enhanced emphysema,cell death, IL-18 production and CXCL13 production in NLRX1 single nullmice exposed to CS was markedly decreased in similarly exposed doublemutant mice (FIGS. 3I-3K, 15, and 16). Overall, these studiesdemonstrate that MAVS plays a critical role in the exaggeratedinflammatory, remodeling, proteolytic, cell death and cytokine responsesthat are induced by CS in states of NLRX1 deficiency.

COPD is a major cause of morbidity and mortality and a major unmetmedical need in the USA and the world (4). In keeping with itsimportance, studies of COPD and models of this disorder have highlightedthe importance of antiviral innate immunity, protease excess, epithelialcell death, and inflammasome activation in disease pathogenesis (5, 6,11, 19-23). The work described herein adds to and integrates thesefindings by demonstrating that mitochondria play an important role inthe regulation of and the effector phase of CS-induced responses.Specifically they demonstrate that NLRX1 is an important inhibitor atbaseline and that CS-stimulates inflammation and remodeling bysuppressing NLRX1 which allows these MAVS-dependent responses to beseen. The inhibition of NLRX1 augmented antiviral innate immuneresponses, protease expression, cell death, and inflammasome activationthereby mechanistically integrating the present and prior observations.The disease relevance of these studies was also confirmed in studiesthat demonstrated that the expression of NLRX1 is decreased in lungsfrom patients with COPD where this suppression correlates with diseaseseverity, abnormal pulmonary function, decreased quality of life andpoor prognosis.

CS is an important stimulator of inflammation and remodeling in avariety of diseases and disorders (3, 24-26). It also exaggeratesantiviral responses in patients with COPD and otherwise healthy smokers(11, 27, 28). The ability of CS and virus/viral PAMPs to synergize inthe generation of inflammation and tissue remodeling is mediated by aMAVS-dependent mechanism (5). The present studies add to theseobservations by demonstrating that MAVS also plays a crucial role in theinflammation and remodeling induced by CS alone. Importantly they alsorevise our understanding of the mechanism that CS uses to engendertissue alterations. Specifically, they demonstrate that optimalCS-induced responses are only seen when CS abrogates the inhibitoryeffects of NLRX1.

NLRX1 is a member of the Nod-like receptor family of intracellularsensors of microbial-and danger-associated molecular patterns (15, 29).In early studies it was identified to be a negative regulator of MAVS, akey adapter molecule of M-RLH antiviral signaling (15). It is now knownto be a negative modulator of LPS-induced TRAF6-NK-κB signaling and hasbeen shown to regulate autophagy (14, 16, 30), bind to UQCRC2 inmitochondrial respiratory chain complex III and regulate mitochondrialproduction of reactive oxidant species (ROS)(31). This effectorrepertoire might have significant implications for the methods describedherein because defects in autophagy/mitophagy lead to the accumulationof damaged, ROS producing, mitochondria which induce inflammasomeactivation (32). In accord with this concept, a recent publicationdemonstrated that MAVS also plays a role as a critical adaptor moleculein inflammasome activation (33). It is contemplated herein thatNLRX1-inhibition of-MAVS-dependent inflammasome activation plays animportant role in the pathogenesis of COPD.

It is demonstrated herein that NLRX1 is suppressed in patients with COPDwhere the degree of suppression correlates with disease severity. It isalso demonstrated that the effects of CS are mediated by anNLRX1-inhibited and MAVS-dependent pathway and that interventions thatabrogate the CS-induced decrease in NLRX1 also abrogate CS-inducedemphysema. These studies demonstrate that the suppression of NLRX1 is acritical event in CS-induced responses. It is also contemplated hereinthat the degree of NLRX1 suppression can be an important index of COPDseverity and that polymorphisms or other alterations that decrease NLRX1function can contribute to disease susceptibility. Importantly, thedemonstration that NLRX1 supplementation abrogates CS-induced alveolardestruction also indicates that interventions that prevent theinhibition of NLRX1, augment the effects of NLRX1, abrogate MAVSfunction or restore NLRX1 suppression can be therapeutically useful inthis disorder.

Methods

Clinical Data.

Three cohorts were used in these studies. In the Yale cohort, freshfrozen lung tissues from 7 controls and 36 patients with COPD wereobtained from the Lung Tissue Research Consortium (LTRC), a nationwideresource program from the National Heart, Lung, and Blood Institute(NHLBI) that provides human lung tissues with highly qualified andextensive phenotype data. This study was approved by the Yale UniversityHuman Investigation Committee and LTRC (#11-99-0005). For the Pittsburghcohort, lung tissues were obtained as previously described (34).

Mouse Models.

All in vivo experiments in animals were approved by the Yale Animal Careand Use Committee (YACUC). The generation and basic characterization ofMAVS null mutant and NLRX1 null mutant mice have been describedpreviously (14, 35). The CS-induced murine emphysema model that wasemployed has been described previously (6, 18).

Laboratory Assessments.

Separation of the cytosol- and mitochondria-enriched fractions wasundertaken using Qproteome mitochondrial isolation kit (Qiagen) as perthe manufacturer's instructions. For the evaluation of lung morphometry,the left lung was inflated with 0.5% low temperature-melting agarose in10% buffered formalin fixative at a constant pressure of 25 cm asdescribed previously (5, 6). Alveolar size was estimated by themeasurement of mean chord length of the air space as describedpreviously (5, 6).

Statistical Analysis.

Comparisons of the levels of NLRX1 in patients with different stages ofCOPD were undertaken with Student's t-test, nonparametric Kruskal-Wallisand Mann-Whitney U tests as appropriate. To evaluate the associationsbetween the levels of NLRX1 expression and clinical variables,nonparametric Pearson correlation analysis was undertaken. For theanalyses of the murine data, groups were compared with Student's t testor with nonparametric Mann-Whitney U test as appropriate. Statisticalsignificance was defined at a level of p≦0.05. All analyses werecompleted with SPSS™ software, version 19.0.

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TABLE 1 Clinical Characteristics of the Study Populations. (a) LTRCCohort COPD^(#) Characteristic^(§) Control GOLD 1 GOLD 2 GOLD 3 GOLD 4Number 7 6 10 10 10 age (yr) 57.7 ± 10.7 73.7 ± 6.9  66.3 ± 9.7  64.2 ±6.9 54.0 ± 7.2  Gender (Male:Female) (2:5) (5:1) (10:0) (7:3) (6:4)Smokers (Non-smokers) 3 (4) 6 10 10 10 Smoking history - pack-yr 15.4 ±19.5 36.5 ± 10.8 54.1 ± 24.6  62.0 ± 25.7 51.8 ± 38.2 FEV₁ (%), Pre-BD93.0 ± 12.1 82.7 ± 10.3 61.6 ± 6.9  32.2 ± 4.2 21.5 ± 7.7  FEV₁ (%),Post BD 98.2 ± 12.4 89.2 ± 6.9  67.7 ± 5.6  34.2 ± 3.3 19.8 ± 2.8 D_(LCO) 96.4 ± 26.6 80.4 ± 16.9 51.75 ± 24.6  31.3 ± 6.3 39.2 ± 16.6 6minute Walking-Distance 413.5 ± 43.8   321. ±, 35.6 318.5 ± 168.9 237.2± 88.9 270.5 ± 69.6  BODE Index 0.1 ± 0.4 1.1 ± 1.1 2.9 ± 2.6  6.8 ± 1.66.8 ± 1.1 SGRQ Score 5.3 ± 9.8 18.1 ± 20.6 36.9 ± 27.7  67.0 ± 10.9 59.2± 15.1 BORG Scale at Termination 0.8 ± 0.3 1.0 ± 1.4 3.3 ± 1.4  4.3 ±1.9 4.9 ± 2.6 NLRX1 mRNA (standardized) 1.22 ± 0.73 1.35 ± 0.39 1.22 ±0.63  0.78 ± 0.29 0.73 ± 0.28 (b) Pittsburgh Cohort Control <1%emphysema 1-10% emphysema >40% emphysema Characteristic (n = 63) (n =20) (n = 51) (n = 26) Age 65.8 ± 10.3 67.9 ± 8.4  68.4 ± 8.4  56.2 ± 7.2Female Gender 27 (43%) 8 (40%) 17 (33%) 16 (62%) Race 2 African-American1 African-American 58 Caucasian 20 Caucasian 50 Caucasian 26 Caucasian 3Other Pack-Years 26.3 ± 34.3 56.1 ± 31.2 65.1 ± 40.0  50.8 ± 25.6 FEV1(% predicted) 97.4 ± 9.2  63.5 ± 12.0 60.3 ± 13.9 24.2 ± 8.2 FVC (%predicted) 93.3 ± 11.8 74.9 ± 12.7 83.4 ± 15.7  56.0 ± 18.7 DLCO (%predicted) 81.9 ± 16.3 70.4 ± 21.8 62.4 ± 16.1 31.5 ± 8.5 HU < −950 (%)0.7 ± 1.1 0.4 ± 0.3 4.2 ± 2.5 48.7 ± 6.9 (c) Asan Cohort CharacteristicControl GOLD 1 GOLD 2 GOLD 3 Number (M:F) 94 (94:0) 45 (45:0) 53 (53:0)1 (1:0) age (mean, STD) 60.6 ± 9.4  68.3 ± 6.1  66.9 ± 6.5  61 Smoker(NS:CS) (94:0) (45:0) (53:0) (1:0) PKYrs (mean, STD) 34.8 ± 17.1 49.5 ±19.4 46.3 ± 24.1 40 FEV1 (%), Pre-BD (mean, STD) 91.0 ± 12.4 83.7 ± 8.3 63.5 ± 7.6  28 FEV1 (%), Post BP (mean, STD) 94.3 ± 12.8 89.2 ± 6.8 68.4 ± 6.7  34 D_(LCO) (mean, STD) 92.9 ± 12.9 78.3 ± 13.9 76.1 ± 14.686 NLRX1 transcriptome (mean, STD) 2.22 ± 1.49 1.69 ± 1.34 1.39 ± 0.871.06 Values are presented as means ± standard deviation (STD) in LTRCYale Cohort (a), Pittsburgh Cohort (b) and Korean Asan cohort,respectively. ^(#)The Global Initiative for Chronic Obstructive LungDisease (GOLD) stage range from stage 1 (GOLD 1) COPD, indicating milddisease, to stage IV (GOLD 4) COPD, indicating very severe disease.^(§)FEV₁(%), pre-BD denotes prebronchodilator FEV₁ (% of predictedvalue). FEV₁(%), post-BD denotes postbronchodilator FEV₁ (% of predictedvalue). D_(LCO) denotes diffusing capacity. The BODE index stands forbody mass index (BMI), obstruction, dyspnea and exercise capacity. TheSGRQ score denotes Saint George Respiratory Questionnaires score. TheBORG scale at termination is the extent of perceived patient exertionthat can be estimated by the Borg scale at the termination of exercise.

Supplemental Methods

Study Populations. Three cohorts were used in these studies. In the Yalecohort, fresh frozen lung tissues from 7 controls and 36 patients withCOPD were obtained from the Lung Tissue Research Consortium (LTRC)(1),as described in the main text. For the Pittsburgh cohort, lung tissueswere obtained as previously described (2). Briefly, controls (n=63) weredefined as the subjects who have no evidence of chronic lung disease aswell as normal pulmonary function tests including lung volumes. Thesubjects with COPD had varying levels of emphysema which were measuredby quantitative CT using HU<-950 as a cutoff. The subjects with COPDwere further stratified by the amount of quantitative emphysema with thethree categories including <1%, which denotes less than 1% emphysema(n=22); 1-10%, which denotes a low level of emphysema between 1 and 10percent (n=45); and the most severe cases, with more than 40% emphysema(n=27). The characteristics of these patients can be seen in Table 1b.For the Asan cohort, subjects were patients who required resection forlung cancer and who were registered in the Asan Biobank. Inclusioncriteria were a FEV1/FVC ratio of less than 0.7 for the COPD group, andnormal spirometry for the control group. This study was approved by theinstitutional review board of Asan Medical Center (#2011-0711) andwritten informed consent was obtained from all patients.

RNA preparation and sequencing for human studies. For the Yale cohort,the mRNAs were extracted from fresh frozen lung tissues from the 7controls and 36 patients with COPD in the NIH-sponsored Lung TissueResearch Consortium (LTRC) cohort. cDNA synthesis and real-timereverse-transcriptase-polymerase-chain-reaction (RT-PCR) assays wereperformed with whole RNA extracted from fresh frozen human lung tissuesusing Bio-Rad kits as per the manufacturer's instructions. The humanNLRX1 primers from Primerbank (3) were utilized for the evaluation ofthe Yale cohort. For the Asan cohort, total RNA was isolated fromapparently normal fresh frozen lung tissue that was remote from the lungcancer. RNA integrity was assessed using an Agilent Bioanalyzer™ and RNApurity was assessed using a NanoDrop™ spectrophotometer. One μg of totalRNA was used to generate cDNA libraries using the TruSeq™ RNA librarykit. The protocol consisted of poly A-selected RNA extraction, RNAfragmentation, reverse transcription using random hexamer primers, and100 bp paired-end sequencing using the Illumina HiSeq™ 2000 system. Thelibraries were quantified using quantitative PCR according to thequantitative PCR Quantification Protocol Guide and qualified using anAgilen Technologies 2100 Bioanalyzer™. For quality control, read qualitywas verified using FastQC™ and read alignment was verified usingPicard™. Differential gene expression (DEG) analysis was performed usingTopHat™ and Cufflinks™ software (4). To estimate expression levels, theRNA-seq reads were mapped to the human genome using TopHat™ (version1.4.1)(5), and quantified using Cufflinks™ software (2.0.0)(6).Cufflinks™ software was run with the UCSC hg19 human genome andtranscriptome references. The numbers of isoform and gene transcriptswere calculated and the relative abundance of transcripts was measuredin fragments per kilobase of exon per million fragments mapped (FPKM)using Cufflinks software. Expression levels were extracted as a FPKMvalue for each gene of each sample using Cufflinks™ software. Genes withFPKM values of 0 across all samples were excluded. Filtered data weresubject to upper quantile normalization. Statistical significance wasdetermined using Student's t-test. The false discovery rate (FDR) wascontrolled by adjusting p values using the Benjamini-Hochberg algorithm.

Laboratory Assessments. Separation of the cytosol- andmitochondria-enriched fractions was undertaken using Qproteome™mitochondrial isolation kit (Qiagen) as per the manufacturer'sinstructions. Immunoblot analyses were undertaken using antibodies forNLRX1 (Proteintech), caspase-1 (Cell Signaling), IL-1β (Santa Cruz),IL-18 (Santa Cruz) and β-actin (Cell Signaling). Immunohistochemistryfor NLRX1 was undertaken to localize the expression of NLRX1. For theevaluation of lung morphometry, subgroups of 5-7 mice were used for theevaluation of mean chord length and the surface area of the lungsfollowing stereological analysis of the lungs according to the ATS/ERSguidelines (7, 8). Briefly, the left lung was inflated with 0.5% lowtemperature-melting agarose in 10% buffered formalin fixative at aconstant pressure of 25 cm as described previously (9, 10). After thefixation, lung volume (V_(L)) of the left lung was determined by thewater immersion method. The images were taken equally spaced andsystematically placed meander-like over the whole surface of the lungsections. Pictures were quantitatively analyzed by using a test systemof points and lines superimposed over the digital images via theSTEPanizer™ program (11). The intersecting points falling on alveolarspace (Pa), alveolar ducts (Pd), and points falling on septum (Ps) werecounted separately among total points (Ptotal). Point counts yieldedrelative volume densities and alveolar surface area (S) was calculatedby the following formula; (1) Airspace fraction(F_(A))=(Pa+Pd+Ps)/Ptotal; (2) Airspace volume (V_(A))=F_(A)×V_(L); (3)S=4V_(A)/Lm (mean linear intercept). In addition, mean chord length ofthe air space was evaluated as described previously by our laboratory(9, 10). Lung tissue lysates were prepared and the levels of tissueCXCL13, IL-1β and IL-18 (R&D Systems) were determined using commercialELISA kits as per the manufacturer's instructions. For the evaluation ofapoptotic cell death/DNA injury, end labeling of exposed 3′-OH ends ofDNA fragments in paraffin embedded tissue was undertaken with the TUNELin situ cell death detection kit AP (Roche Diagnostics) using theinstructions provided by the manufacturer. To overexpress NLRX1 gene invivo, the gene was integrated into lentiviral vector. The lentiviralvector backbones used in this study were purchased from SystemBiosciences Inc. (SBI, CA, USA, Catalog #CD511B-1). The NLRX1 cDNA clonewas purchased from Origene (Origene, Md., USA, Catalog #MC204753).pPACKH1-XL HIV Lentivector Packaging Kit (SBI, CA, USA, Catalog#LV510A-1) and virus precipitation solution (SBI, CA, USA, Catalog#LV810A-1) were used to package the lentiviral vectors, according to themanufacturer's instructions. The lentiviral titer kit (SBI, CA, USA,Catalog #LV961A-1) was used to determine the amount of viral vectorsaccording to the manufacturer's instructions. Intranasal administrationof either the lenti-NLRX1, or lenti-control vectors was performed on6-wk-old C57BL/6J male mice. The amount of 1×10⁸ TU of lenti-NLRX1 orlenti-control vectors per mouse was administered. CS exposure experimentwas started from 2 weeks after intranasal treatment.

REFERENCES

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What is claimed herein is:
 1. A method of treating COPD, cigarettesmoke-induced lung damage, or emphysema in a subject in need thereof,the method comprising administering an inhibitor of MAVS.
 2. The methodof claim 1, wherein the cigarette smoke-induced lung damage is selectedfrom the group consisting of: inflammation; alveolar destruction;protease induction; structural cell apoptosis; and inflammasomeactivation.
 3. The method of claim 1, wherein the emphysema is cigarettesmoke induced emphysema.
 4. The method of claim 1, wherein the inhibitorof MAVS is an agonist of NLRX1.
 5. The method of claim 1, wherein theinhibitor of MAVS is a modulator of NLRX1-dependent pathways.
 6. Themethod of claim 4, wherein the agonist of NLRX1 is a nucleic acidencoding NLRX1 or an NLRX1 polypeptide.
 7. The method of claim 1,wherein the subject is a subject determined to have a decreased level ofNLRX1 expression.
 8. A method of treatment for COPD, emphysema, and/orcigarette-induced lung damage comprising; measuring the level of NLRX1in a test sample obtained from a subject; treating the subject when thelevel of NLRX1 is decreased relative to a reference level.
 9. The methodof claim 8, wherein the treatment comprises a treatment selected fromthe group consisting of: administration of a bronchodilator;administration of an inhaled steroid; administration of oxygen therapy;bullectomy; lung volume reduction surgery; smoking cessation; lungtransplant; administration of an inhibitor of MAVS; administration of anagonist of NLRX1; administration of a nucleic acid encoding NLRX1; andadministration of a NLRX1 polypeptide.
 10. The method of claim 8,wherein the level of NLRX1 is determined by measuring the level of anucleic acid.
 11. The method of claim 10, wherein the level of NLRX1 isdetermined by measuring the level of NLRX1 RNA transcript.
 12. Themethod of claim 10, wherein the level of the nucleic acid is determinedusing a method selected from the group consisting of: RT-PCR;quantitative RT-PCR; Northern blot; microarray based expressionanalysis; next-generation sequencing; and RNA in situ hybridization. 13.The method of claim 8, wherein the level of NLRX1 is determined bymeasuring the level of NLRX1 polypeptide.
 14. The method of claim 13,wherein the level of the polypeptide is determined using a methodselected from the group consisting of: Western blot;immunoprecipitation; enzyme-linked immunosorbent assay (ELISA);radioimmunological assay (RIA); sandwich assay; fluorescence in situhybridization (FISH); immunohistological staining; radioimmunometricassay; immunofluoresence assay; mass spectroscopy; FACS; andimmunoelectrophoresis assay.
 15. The method of claim 13, wherein thepolypeptide level is measured using immunochemistry.
 16. The method ofclaim 15, wherein the antibody reagent is detectably labeled orgenerates a detectable signal.
 17. The method of claim 8, wherein theexpression level of NLRX1 is normalized relative to the expression levelof one or more reference genes or reference proteins.
 18. The method ofclaim 17, wherein the reference level of NLRX1 is the expression levelof NLRX1 in a prior sample obtained from the subject.
 19. The method ofclaim 8, wherein the subject is a subject with a history of smoking. 20.The method of claim 8, wherein the sample is selected from the groupconsisting of: a lung biopsy; bronchoalveolar lavage (BAL); sputum;induced sputum; blood; plasma; and serum.